mecB

ABSTRACT

The invention provides mecB polypeptides and polynucleotides encoding mecB polypeptides and methods for producing such polypeptides by recombinant techniques. Also provided are methods for utilizing mecB polypeptides to screen for antibacterial compounds.

RELATED APPLICATIONS

This application claims benefit of U.S. Provisional Patent ApplicationSer. No. 60/057,535, filed Sep. 4, 1997.

FIELD OF THE INVENTION

This invention relates to newly identified polynucleotides andpolypeptides, and their production and uses, as well as their variants,agonists and antagonists, and their uses. In particular, the inventionrelates to novel polynucleotides and polypeptides of the ClpC ATPasefamily, hereinafter referred to as "mecB".

BACKGROUND OF THE INVENTION

It is particularly preferred to employ Staphylococcal genes and geneproducts as targets for the development of antibiotics. TheStaphylococci make up a medically important genera of microbes. They areknown to produce two types of disease, invasive and toxigenic. Invasiveinfections are characterized generally by abscess formation effectingboth skin surfaces and deep tissues. S. aureus is the second leadingcause of bacteremia in cancer patients. Osteomyelitis, septic arthritis,septic thrombophlebitis and acute bacterial endocarditis are alsorelatively common. There are at least three clinical conditionsresulting from the toxigenic properties of Staphylococci. Themanifestation of these diseases result from the actions of exotoxins asopposed to tissue invasion and bacteremia. These conditions include:Staphylococcal food poisoning, scalded skin syndrome and toxic shocksyndrome.

The frequency of Staphylococcus aureus infections has risen dramaticallyin the past few decades. This has been attributed to the emergence ofmultiply antibiotic resistant strains and an increasing population ofpeople with weakened immune systems. It is no longer uncommon to isolateStaphylococcus aureus strains which are resistant to some or all of thestandard antibiotics. This phenomenon has created a demand for both newanti-microbial agents, vaccines, and diagnostic tests for this organism.

Clearly, there exists a need for factors, such as the mecB embodimentsof the invention, that have a present benefit of being useful to screencompounds for antibiotic activity. Such factors are also useful todetermine their role in pathogenesis of infection, dysfunction anddisease. There is also a need for identification and characterization ofsuch factors and their antagonists and agonists to find ways to prevent,ameliorate or correct such infection, dysfunction and disease.

Certain of the polypeptides of the invention possess amino acid sequencehomology to a known ClpC protein.

SUMMARY OF THE INVENTION

It is an object of the invention to provide polypeptides that have beenidentified as novel mecB polypeptides by homology between the amino acidsequence set out in Table 1 [SEQ ID NO: 2 or 4] and a known amino acidsequence or sequences of other proteins such as ClpC protein.

It is a further object of the invention to provide polynucleotides thatencode mecB polypeptides, particularly polynucleotides that encode thepolypeptide herein designated mecB.

In a particularly preferred embodiment of the invention thepolynucleotide comprises a region encoding mecB polypeptides comprisinga sequence set out in Table 1 [SEQ ID NO: 1 or 3] which includes a fulllength gene, or a variant thereof.

In another particularly preferred embodiment of the invention there is anovel mecB protein from Staphylococcus aureus comprising the amino acidsequence of Table 1 [SEQ ID NO:2 or 4], or a variant thereof.

A further aspect of the invention there are provided isolated nucleicacid molecules encoding mecB, particularly Staphylococcus aureus mecB,including mRNAs, cDNAs, genomic DNAs. Further embodiments of theinvention include biologically, diagnostically, prophylactically,clinically or therapeutically useful variants thereof, and compositionscomprising the same.

In accordance with another aspect of the invention, there is providedthe use of a polynucleotide of the invention for therapeutic orprophylactic purposes, in particular genetic immunization. Among theparticularly preferred embodiments of the invention are naturallyoccurring allelic variants of mecB and polypeptides encoded thereby.

Another aspect of the invention there are provided novel polypeptides ofStaphylococcus aureus referred to herein as mecB as well asbiologically, diagnostically, prophylactically, clinically ortherapeutically useful variants thereof, and compositions comprising thesame.

Among the particularly preferred embodiments of the invention arevariants of mecB polypeptide encoded by naturally occurring alleles ofthe mecB gene.

In a preferred embodiment of the invention there are provided methodsfor producing the aforementioned mecB polypeptides.

In accordance with yet another aspect of the invention, there areprovided inhibitors to such polypeptides, useful as antibacterialagents, including, for example, antibodies.

In accordance with certain preferred embodiments of the invention, thereare provided products, compositions and methods for assessing mecBexpression, treating disease, assaying genetic variation, andadministering a mecB polypeptide or polynucleotide to an organism toraise an immunological response against a bacteria, especially aStaphylococcus aureus bacteria.

In accordance with certain preferred embodiments of this and otheraspects of the invention there are provided polynucleotides thathybridize to mecB polynucleotide sequences, particularly under stringentconditions.

In certain preferred embodiments of the invention there are providedantibodies against mecB polypeptides.

In other embodiments of the invention there are provided methods foridentifying compounds which bind to or otherwise interact with andinhibit or activate an activity of a polypeptide or polynucleotide ofthe invention comprising: contacting a polypeptide or polynucleotide ofthe invention with a compound to be screened under conditions to permitbinding to or other interaction between the compound and the polypeptideor polynucleotide to assess the binding to or other interaction with thecompound, such binding or interaction being associated with a secondcomponent capable of providing a detectable signal in response to thebinding or interaction of the polypeptide or polynucleotide with thecompound; and determining whether the compound binds to or otherwiseinteracts with and activates or inhibits an activity of the polypeptideor polynucleotide by detecting the presence or absence of a signalgenerated from the binding or interaction of the compound with thepolypeptide or polynucleotide.

In accordance with yet another aspect of the invention, there areprovided mecB agonists and antagonists, preferably bacteriostatic orbacteriocidal agonists and antagonists.

In a further aspect of the invention there are provided compositionscomprising a mecB polynucleotide or a mecB polypeptide foradministration to a cell or to a multicellular organism.

Various changes and modifications within the spirit and scope of thedisclosed invention will become readily apparent to those skilled in theart from reading the following descriptions and from reading the otherparts of the present disclosure.

DESCRIPTION OF THE INVENTION

The invention relates to novel mecB polypeptides and polynucleotides asdescribed in greater detail below. In particular, the invention relatesto polypeptides and polynucleotides of a novel mecB of Staphylococcusaureus, which is related by amino acid sequence homology to ClpCpolypeptide. The invention relates especially to mecB having thenucleotide and amino acid sequences set out in Table 1 as SEQ ID NO: 1and SEQ ID NO: 2 respectively.

                                      TABLE 1                                     __________________________________________________________________________    mecB Polynucleotide and Polypeptide Sequences                                 __________________________________________________________________________    (A) Sequences from Staphylococcus aureus mecB polynucleotide                  sequence [SEQ ID NO:1].                                                       5'-                                                                              1                                                                              ATGTTATTTG GTAGATTAAC TGAACGTGCA CAGCGCGTAT TAGCACATGC                      51                                                                              ACAAGAAGAA GCAATTCGTT TAAATCATTC TAATATAGGA ACAGAACACC                     101                                                                              TATTATTGGG GTTAATGAAA GAACCTGAAG GAATTGCTGC AAAAGTATTA                     151                                                                              GAAAGTTTTA ATATCACTGA AGATAAAGTA ATTGAAGAAG TTGAAAAATT                     201                                                                              AATCGGACAT GGTCAAGATC ATGTTGGTAC ATTGCATTAT ACACCTAGAG                     251                                                                              CTAAAAAAGT CATTGAATTA TCGATGGATG AAGCTAGAAA ATTACATCAC                     301                                                                              AATTTTGTTG GAACGGTTCA TCTTTTATTA GGCTTGATTC GTGAAAATGA                     351                                                                              AGGTGTTGCA GCAAGAGTTT TTGCAAATCT AGATTTAAAT ATTACTAAAG                     401                                                                              CGCGTGCACG GGTTGTGAAA GCTTTAGGAA ACCCTGAAAT GAGTAATAAA                     451                                                                              AATGCACAAG CTAGTAAGTC AAATAATACT CCAACTTTAG ATAGTTTAGC                     501                                                                              TCGTGACTTA ACAGTCATTG CCAAAGACGG TACATTAGAT CCTGTTATAG                     551                                                                              GACGTGATAA AGAAATTACA CGTGTAATTG AAGTATTAAG TAGACGTACG                     601                                                                              AAAAACAATC CTGTACTTAT TGGAGAGCCA GGTGTTGGTA AAACTGCTAT                     651                                                                              TGCTGAAGGT TTAGCGCAAG CCATAGTGAA TAATGAGGTA CCAGAGACAT                     701                                                                              TAAAAGATAA GCGTGTTATG TCTTTAGATA TGGGAACAGT AGTTGCAGGT                     751                                                                              ACTAAATATC GTGGTGAATT TGAAGAGCGT CTGAAAAAGG TTATGGAAGA                     801                                                                              AATCCAACAA GCAGGTAATG TCATCCTATT TATTGATGAG TTGCATACTT                     851                                                                              TAGTTGGTGC TGGTGGTGCT GAAGGTGCTA TCGATGCTTC GAATATTTTG                     901                                                                              AAACCGGCAT TAGCACGTGG TGAATTACAA TGTATTGGTG CTACTACATT                     951                                                                              AGATGAATAT CGCAAAAATA TTGAAAAAGA CGCGGCTTTA GAACGTCGTT                    1001                                                                              TCCAACCTGT ACAAGTTGAT GAACCTTCAG TAGTAGATAC AGTTGCTATT                    1051                                                                              TTAAAAGGAT TAAGAGATCG TTACGAAGCA CACCATCGTA TTAATATTTC                    1101                                                                              AGACGAAGCT ATTGAAGCAG CTGTTAAATT AAGTAACAGA TACGTTTCAG                    1151                                                                              ATCGTTTCTT ACCAGATAAA GCAATTGATT TAATTGATGA AGCAAGTTCT                    1201                                                                              AAAGTAAGAC TTAAGAGTCA TACGACACCT AATAATTTAA AAGAAATTGA                    1251                                                                              ACAAGAAATT GAAAAAGTTA AAAATGAAAA AGATGCCGCA GTACATGCTC                    1301                                                                              AAGAGTTTGA AAATGCTGCT AACCTGCGTG ATAAACAAAC AAAACTTGAA                    1351                                                                              AAGCAATATG AAGAAGCTAA AAATGAATGG AAGAATGCAC AAAATGGCAT                    1401                                                                              GTCAACTTCA TTGTCAGAAG AAGATATTGC TGAAGTTATT GCAGGATGGA                    1451                                                                              CAGGTATCCC ATTAACTAAA ATCAATGAAA CAGAATCTGA AAAACTTCTT                    1501                                                                              AGTCTAGAAG ATACATTACA TGAGAGAGTT ATTGGGCAAA AAGATGCTGT                    1551                                                                              TAATTCAATC AGTAAAGCGG TTAGACGTGC CCGTGCAGGG TTAAAAGATC                    1601                                                                              CTAAACGACC AATTGGTAGC TTTATCTTCC TTGGACCAAC TGGTGTTGGT                    1651                                                                              AAAACTGAAT TAGCTAGAGC TTTAGCTGAA TCAATGTTTG GCGATGATGA                    1701                                                                              TGCGATGATC CGTGTAGACA TGAGTGAATT TATGGAAAAA CACGCAGTGA                    1751                                                                              GCCGATTAGT TGGTGCTCCT CCAGGATATG TTGGTCATGA TGATGGTGGA                    1801                                                                              CAATTAACTG AAAAAGTTAG ACGTAAACCA TATTCTGTAA TTTTATTTGA                    1851                                                                              TGAAATTGAA AAAGCTCATC CAGATGTATT TAATATTCTA TTACAAGTTT                    1901                                                                              TAGATGATGG ACATTTGACA GATACAAAAG GACGTACAGT TGATTTCAGA                    1951                                                                              AATACAATTA TCATAATGAC ATCAAACGTT GGGGCACAAG AATTACAAGA                    2001                                                                              TCAACGATTT GCTGGATTCG GTGGTTCAAG TGATGGACAA GATTATGAAA                    2051                                                                              CAATTCGAAA AACGATGTTA AAAGAATTAA AAAATTCATT CCGTCCAGAA                    2101                                                                              TTTTTAAACC GTGTAGATGA TATCATTGTA TTCCATAAAC TAACAAAAGA                    2151                                                                              AGAATTAAAA GAAATTGTAA CAATGATGGT TAATAAATTA ACAAATCGAT                    2201                                                                              TATCTGAACA AAACATAAAT ATTATTGTTA CTGATAAAGC GAAAGACAAA                    2251                                                                              ATCGCAGAAG AAGGATATGA TCCAGAATAT GGTGCAAGAC CATTAATTAG                    2301                                                                              AGCGATACAA AAAACTATCG AAGATAATTT AAGTGAATTA ATATTAGATG                    2351                                                                              GTAATCAAAT TGAAGGTAAG AAAGTTACAG TAGATCATGA TGGTAAAGAG                    2401                                                                              TTTAAATATG ACATTGCTGA ACAAACTTCA GAAACTAAAA CACCATCGCA                    2451                                                                              AGCATAATTA TAAAACAGTC CAAAACAAAT TAAAGTTTTG GGCTGTTTTT                    2501                                                                              TTAGTAGCAT TGAACTATAG AAATTCGTGA AAGTATCCAT CAACGAAACA                    2551                                                                              ATCTAATAAA ACAATCATCA AAGGATAGTT AAGAATTATA TGTAACAAG-3'                  (B) Staphylococcus aureus mecB polypeptide sequence                           deduced from the polynucleotide sequence in this                              table [SEQ ID NO:2].                                                          NH.sub.2 -                                                                       1                                                                              MLFGRLTERA QRVLAHAQEE AIRLNHSNIG TEHLLLGLMK EPEGIAAKVL                      51                                                                              ESFNITEDKV IEEVEKLJGH GQDHVGTLHY TPRAKKVTEL SMDEARKLHH                     101                                                                              NFVGTVHLLL GLIRENEGVA ARVFANLDLN JTKARARVVK ALGNPEMSNK                     151                                                                              NAQASKSNNT PTLDSLARDL TVIAKDGTLD PVIGRDKEIT RVIEVLSRRT                     201                                                                              KNNPVLIGEP GVGKTAIAEG LAQAIVNNEV PETLKDKRVM SLDMGTVVAG                     251                                                                              TKYRGEFEER LKKVMEEIQQ AGNVILFIDE LHTLVGAGGA EGAIDASNIL                     301                                                                              KPALARGELQ CIGATTLDEY RKNIEKDAAL ERRFQPVQVD EPSVVDTVAI                     351                                                                              LKGLRDRYEA HHRJNISDEA IEAAVKLSNR YVSDRFLPDK AIDLIDEASS                     401                                                                              KVRLKSHTTP NNLKEIEQEI EKVKNEKDAA VHAQEFENAA NLRDKQTKLE                     451                                                                              KQYEEAKNEW KNAQNGMSTS LSEEDIAEVI AGWTGIPLTK INETESEKLL                     501                                                                              SLEDTLHERV IGQKDAVNSI SKAVRRARAG LKDPKRPIGS FIFLGPTGVG                     551                                                                              KTELARALAE SMFGDDDAMI RVDMSEFMEK HAVSRLVGAP PGYVGHDDGG                     601                                                                              QLTEKVRRKP YSVILFDEIE KAHPDVFNIL LQVLDDGHLT DTKGRTVDFR                     651                                                                              NTIIIMTSNV GAQELQDQRF AGFGGSSDGQ DYETIRKTML KELKNSFRPE                     701                                                                              FLNRVDDIIV FHKLTKEELK EIVTMMVNKL TNRLSEQNIN IIVTDKAKDK                     751                                                                              IAEEGYDPEY GARPLIRAIQ KTIEDNLSEL ILDGNQIEGK KVTVDHDGKE                     801                                                                              FKYDIAEQTS ETKTPSQA*L *NSPKQIKVL GCFFSSIEL* KFVKVSINET                     851                                                                              I**NNHQRIV KNYM*Q                                                         -COOH                                                                         (C) Polynucleotide sequences comprising Staphylococcus                        aureus mecB ORF sequence [SEQ ID NO:3].                                       5'-                                                                              1                                                                              ATGAGTAATA AAAATGCACA AGCTAGTAAG TCAAATAATA CTCCAACTTT                      51                                                                              AGATAGTTTA GCTCGTGACT TAACAGTCAT TGCCAAAGAC GGTACATTAG                     101                                                                              ATCCTGTTAT AGGACGTGAT AAAGAAATTA CACGTGTAAT TGAAGTATTA                     151                                                                              AGTAGACGTA CGAAAAACAA TCCTGTACTT ATTGGAGAGC CAGGTGTTGG                     201                                                                              TAAAACTGCT ATTGCTGAAG GTTTAGCGCA AGCCATAGTG AATAATGAGG                     251                                                                              TACCAGAGAC ATTAAAAGAT AAGCGTGTTA TGTCTTTAGA TATGGGAACA                     301                                                                              GTAGTTGCAG GTACTAAATA TCGTGGTGAA TTTGAAGAGC GTCTGAAAAA                     351                                                                              GGTTATGGAA GAAATCCAAC AAGCAGGTAA TGTCATCCTA TTTATTGATG                     401                                                                              AGTTGCATAC TTTAGTTGGT GCTGGTGGTG CTGAAGGTGC TATCGATGCT                     451                                                                              TCGAATATTT TGAAACCGGC ATTAGCACGT GGTGAATTAC AATGTATTGG                     501                                                                              TGCTACTACA TTAGATGAAT ATCGCAAAAA TATTGAAAAA GACGCGGCTT                     551                                                                              TAGAACGTCG TTTCCAACCT GTACAAGTTG ATGAACCTTC AGTAGTAGAT                     601                                                                              ACAGTTGCTA TTTTAAAAGG ATTAAGAGAT CGTTACGAAG CACACCATCG                     651                                                                              TATTAATATT TCAGACGAAG CTATTGAAGC AGCTGTTAAA TTAAGTAACA                     701                                                                              GATACGTTTC AGATCGTTTC TTACCAGATA AAGCAATTGA TTTAATTGAT                     751                                                                              GAAGCAAGTT CTAAAGTAAG ACTTAAGAGT CATACGACAC CTAATAATTT                     801                                                                              AAAAGAAATT GAACAAGAAA TTGAAAAAGT TAAAAATGAA AAAGATGCCG                     851                                                                              CAGTACATGC TCAAGAGTTT GAAAATGCTG CTAACCTGCG TGATAAACAA                     901                                                                              ACAAAACTTG AAAAGCAATA TGAAGAAGCT AAAAATGAAT GGAAGAATGC                     951                                                                              ACAAAATGGC ATGTCAACTT CATTGTCAGA AGAAGATATT GCTGAAGTTA                    1001                                                                              TTGCAGGATG GACAGGTATC CCATTAACTA AAATCAATGA AACAGAATCT                    1051                                                                              GAAAAACTTC TTAGTCTAGA AGATACATTA CATGAGAGAG TTATTGGGCA                    1101                                                                              AAAAGATGCT GTTAATTCAA TCAGTAAAGC GGTTAGACGT GCCCGTGCAG                    1151                                                                              GGTTAAAAGA TCCTAAACGA CCAATTGGTA GCTTTATCTT CCTTGGACCA                    1201                                                                              ACTGGTGTTG CTAAAACTGA ATTAGCTAGA GCTTTAGCTG AATCAATGTT                    1251                                                                              TGGCGATGAT GATGCGATGA TCCGTGTAGA CATGAGTGAA TTTATGGAAA                    1301                                                                              AACACGCAGT GAGCCGATTA GTTGGTGCTC CTCCAGGATA TGTTGGTCAT                    1351                                                                              GATGATGGTG GACAATTAAC TGAAAAAGTT AGACGTAAAC CATATTCTGT                    1401                                                                              AATTTTATTT GATCAAATTG AAAAAGCTCA TCCAGATGTA TTTAATATTC                    1451                                                                              TATTACAAGT TTTAGATGAT GGACATTTGA CAGATACAAA AGGACGTACA                    1501                                                                              GTTGATTTCA GAAATACAAT TATCATAATG ACATCAAACG TTGGGGCACA                    1551                                                                              AGAATTACAA GATCAACGAT TTGCTGGATT CGGTGGTTCA AGTGATGGAC                    1601                                                                              AAGATTATGA AACAATTCGA AAAACGATGT TAAAAGAATT AAAAAATTCA                    1651                                                                              TTCCGTCCAG AATTTTTAAA CCGTGTAGAT GATATCATTG TATTCCATAA                    1701                                                                              ACTAACAAAA GAAGAATTAA AAGAAATTGT AACAATGATG GTTAATAAAT                    1751                                                                              TAACAAATCG ATTATCTGAA CAAAACATAA ATATTATTGT TACTGATAAA                    1801                                                                              GCGAAAGACA AAATCGCAGA AGAAGGATAT GATCCAGAAT ATGGTGCAAG                    1851                                                                              ACCATTAATT AGAGCGATAC AAAAAACTAT CGAAGATAAT TTAAGTGAAT                    1901                                                                              TAATATTAGA TGGTAATCAA ATTGAAGGTA AGAAAGTTAC AGTAGATCAT                    1951                                                                              GATGGTAAAG AGTTTAAATA TGACATTGCT GAACAAACTT CAGAAACTAA                    2001                                                                              AACACCATCG CAACCATAA                                                      -3'                                                                           (D) Staphylococcus aureus mecB polypeptide sequence                           deduced from the polynucleotide ORF sequence in this                          table [SEQ ID NO:4].                                                          NH.sub.2 -                                                                       1                                                                              MSNKNAQASK SNNTPTLDSL ARDLTVIAKD GTLDPVIGRD KEITRVIEVL                      51                                                                              SRRTKNNPVL IGEPGVGKTA IAEGLAQAJV NNEVPETLKD KRVMSLDMGT                     101                                                                              VVAGTKYRGE FEERLKKVME EIQQAGNVIL FIDELHTLVG AGGAEGAIDA                     151                                                                              SNILKPALAR GELQCIGATT LDEYRKNIEK DAALERRFQP VQVDEPSVVD                     201                                                                              TVAILKGLRD RYEAHHRINI SDEAIEAAVK LSNRYVSDRF LPDKAIDLID                     251                                                                              EASSKVRLKS HTTPNNLKEI EQEIEKVKNE KDAAVHAQEF ENAANLRDKQ                     301                                                                              TKLEKQYEEA KNEWKNAQNG MSTSLSEEDI AEVIAGWTGI PLTKINETES                     351                                                                              EKLLSLEDTL HERVIGQKDA VNSISKAVRR ARAGLKDPKR PIGSFIFLGP                     401                                                                              TCVGKTELAR ALAESMFGDD DAMIRVDMSE FMEKHAVSRL VGAPPGYVGH                     451                                                                              DDGGQLTEKV RRKPYSVILF DEIEKAHPDV FNILLQVLDD GHLTDTKGRT                     501                                                                              VDFRNTIIIM TSNVGAQELQ DQRFAGFGGS SDGQDYETIR KTMLKELKNS                     551                                                                              FRPEFLNRVD DIIVFHKLTK EELKEIVTMM VNKLTNRLSE QNINIIVTDK                     601                                                                              AKDKIAEEGY DPEYGARPLI PAIQKTIEDN LSELJLDGNQ IEGKKVTVDH                     651                                                                              DGKEFKYDIA EQTSETKTPS QA                                                  -COOH                                                                         __________________________________________________________________________

Deposited Materials

A deposit containing a Staphylococcus aureus WCUH 29 strain has beendeposited with the National Collections of Industrial and MarineBacteria Ltd. (herein "NCIMB"), 23 St. Machar Drive, Aberdeen AB2 1RY,Scotland on Sep. 11, 1995 and assigned NCIMB Deposit No. 40771, andreferred to as Staphylococcus aureus WCUH29 on deposit. TheStaphylococcus aureus strain deposit is referred to herein as "thedeposited strain" or as "the DNA of the deposited strain."

The deposited strain contains the full length mecB gene. The sequence ofthe polynucleotides contained in the deposited strain, as well as theamino acid sequence of the polypeptide encoded thereby, are controllingin the event of any conflict with any description of sequences herein.

The deposit of the deposited strain has been made under the terms of theBudapest Treaty on the International Recognition of the Deposit ofMicro-organisms for Purposes of Patent Procedure. The strain will beirrevocably and without restriction or condition released to the publicupon the issuance of a patent. The deposited strain is provided merelyas convenience to those of skill in the art and is not an admission thata deposit is required for enablement, such as that required under 35U.S.C. §112.

A license may be required to make, use or sell the deposited strain, andcompounds derived therefrom, and no such license is hereby granted.

One aspect of the invention there is provided an isolated nucleic acidmolecule encoding a mature polypeptide expressible by the Staphylococcusaureus WCUH 29 strain contained in the deposited strain. Furtherprovided by the invention are mecB nucleotide sequences of the DNA inthe deposited strain and amino acid sequences encoded thereby. Alsoprovided by the invention are mecB polypeptide sequences isolated fromthe deposited strain and amino acid sequences derived therefrom.

Polypeptides

The polypeptides of the invention include a polypeptide of Table 1 [SEQID NO:2 or 4] (in particular the mature polypeptide) as well aspolypeptides and fragments, particularly those which have the biologicalactivity of mecB, and also those which have at least 70% identity to apolypeptide of Table 1 [SEQ ID NO:1 or 3] or the relevant portion,preferably at least 80% identity to a polypeptide of Table 1 [SEQ IDNO:2 or 4 and more preferably at least 90% similarity (more preferablyat least 90% identity) to a polypeptide of Table 1 [SEQ ID NO:2 or 4]and still more preferably at least 95% similarity (still more preferablyat least 95% identity) to a polypeptide of Table 1 [SEQ ID NO:2 or 4]and also include portions of such polypeptides with such portion of thepolypeptide generally containing at least 30 amino acids and morepreferably at least 50 amino acids.

The invention also includes polypeptides of the formula:

    X--(R.sub.1).sub.m --(R.sub.2)--(R.sub.3).sub.n --Y

wherein, at the amino terminus, X is hydrogen, and at the carboxylterminus, Y is hydrogen or a metal, R₁ and R₃ are any amino acidresidue, m is an integer between 1 and 1000 or zero, n is an integerbetween 1 and 1000 or zero, and R₂ is an amino acid sequence of theinvention, particularly an amino acid sequence selected from Table 1. Inthe formula above R₂ is oriented so that its amino terminal residue isat the left, bound to R₁, and its carboxy terminal residue is at theright, bound to R₃. Any stretch of amino acid residues denoted by eitherR group, where m and/or n is greater than 1, may be either aheteropolymer or a homopolymer, preferably a heteropolymer. A fragmentis a variant polypeptide having an amino acid sequence that entirely isthe same as part but not all of the amino acid sequence of theaforementioned polypeptides. As with mecB polypeptides fragments may be"free-standing," or comprised within a larger polypeptide of which theyform a part or region, most preferably as a single continuous region, asingle larger polypeptide.

Preferred fragments include, for example, truncation polypeptides havinga portion of an amino acid sequence of Table 1 [SEQ ID NO:2 or 4], or ofvariants thereof, such as a continuous series of residues that includesthe amino terminus, or a continuous series of residues that includes thecarboxyl terminus. Degradation forms of the polypeptides of theinvention in a host cell, particularly a Staphylococcus aureus, are alsopreferred. Further preferred are fragments characterized by structuralor functional attributes such as fragments that comprise alpha-helix andalpha-helix forming regions, beta-sheet and beta-sheet-forming regions,turn and turn-forming regions, coil and coil-forming regions,hydrophilic regions, hydrophobic regions, alpha amphipathic regions,beta amphipathic regions, flexible regions, surface-forming regions,substrate binding region, and high antigenic index regions.

Also preferred are biologically active fragments which are thosefragments that mediate activities of mecB, including those with asimilar activity or an improved activity, or with a decreasedundesirable activity. Also included are those fragments that areantigenic or immunogenic in an animal, especially in a human.Particularly preferred are fragments comprising receptors or domains ofenzymes that confer a function essential for viability of Staphylococcusaureus or the ability to initiate, or maintain cause disease in anindividual, particularly a human.

Variants that are fragments of the polypeptides of the invention may beemployed for producing the corresponding full-length polypeptide bypeptide synthesis; therefore, these variants may be employed asintermediates for producing the fill-length polypeptides of theinvention.

In addition to the standard single and triple letter representations foramino acids, the term "X" or "Xaa" may also be used in describingcertain polypeptides of the invention. "X" and "Xaa" mean that any ofthe twenty naturally occuring amino acids may appear at such adesignated position in the polypeptide sequence.

Polynucleotides

Another aspect of the invention relates to isolated polynucleotides,including the fill length gene, that encode the mecB polypeptide havinga deduced amino acid sequence of Table 1 [SEQ ID NO:2 or 4] andpolynucleotides closely related thereto and variants thereof.

Using the information provided herein, such as a polynucleotide sequenceset out in Table 1 [SEQ ID NO:1 or 3], a polynucleotide of the inventionencoding mecB polypeptide may be obtained using standard cloning andscreening methods, such as those for cloning and sequencing chromosomalDNA fragments from bacteria using Staphylococcus aureus WCUH 29 cells asstarting material, followed by obtaining a full length clone. Forexample, to obtain a polynucleotide sequence of the invention, such as asequence given in Table 1 [SEQ ID NO:1 or 3], typically a library ofclones of chromosomal DNA of Staphylococcus aureus WCUH 29 in E.coli orsome other suitable host is probed with a radiolabeled oligonucleotide,preferably a 17-mer or longer, derived from a partial sequence. Clonescarrying DNA identical to that of the probe can then be distinguishedusing stringent conditions. By sequencing the individual clones thusidentified with sequencing primers designed from the original sequenceit is then possible to extend the sequence in both directions todetermine the full gene sequence. Conveniently, such sequencing isperformed using denatured double stranded DNA prepared from a plasmidclone. Suitable techniques are described by Maniatis, T., Fritsch, E. F.and Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, 2nd Ed.;Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989).(see in particular Screening By Hybridization 1.90 and SequencingDenatured Double-Stranded DNA Templates 13.70). Illustrative of theinvention, the polynucleotide set out in Table 1 [SEQ ID NO:1 or 3] wasdiscovered in a DNA library derived from Staphylococcus aureus WCUH 29.

The DNA sequence set out in Table 1 [SEQ ID NO: 1 or 3] contains an openreading frame encoding a protein having about the number of amino acidresidues set forth in Table 1 [SEQ ID NO:2 or 4] with a deducedmolecular weight that can be calculated using amino acid residuemolecular weight values well known in the art. The polynucleotide of SEQID NO: 1, between nucleotide number 1 and the stop codon which begins atnucleotide number 2455 of SEQ ID NO: 1, encodes the polypeptide of SEQID NO:2.

MecB of the invention is structurally related to other proteins of theClpC ATPase family.

The invention provides a polynucleotide sequence identical over itsentire length to a coding sequence in Table 1 [SEQ ID NO: 1 or 3]. Alsoprovided by the invention is the coding sequence for the maturepolypeptide or a fragment thereof, by itself as well as the codingsequence for the mature polypeptide or a fragment in reading frame withother coding sequence, such as those encoding a leader or secretorysequence, a pre-, or pro- or prepro- protein sequence. Thepolynucleotide may also contain non-coding sequences, including forexample, but not limited to non-coding 5' and 3' sequences, such as thetranscribed, non-translated sequences, termination signals, ribosomebinding sites, sequences that stabilize mRNA, introns, polyadenylationsignals, and additional coding sequence which encode additional aminoacids. For example, a marker sequence that facilitates purification ofthe fused polypeptide can be encoded. In certain embodiments of theinvention, the marker sequence is a hexa-histidine peptide, as providedin the pQE vector (Qiagen, Inc.) and described in Gentz et al., Proc.Natl. Acad. Sci., USA 86: 821-824 (1989), or an HA tag (Wilson et al.,Cell 37: 767 (1984). Polynucleotides of the invention also include, butare not limited to, polynucleotides comprising a structural gene and itsnaturally associated sequences that control gene expression.

A preferred embodiment of the invention is a polynucleotide ofcomprising nucleotide 1 to the nucleotide immediately upstream of orincluding nucleotide 2455 set forth in SEQ ID NO: 1 of Table 1, both ofwhich encode the mecB polypeptide.

The invention also includes polynucleotides of the formula:

    X--(R.sub.1).sub.m --(R.sub.2)--(R.sub.3).sub.n --Y

wherein, at the 5' end of the molecule, X is hydrogen or together with Ydefines a covalent bond, and at the 3' end of the molecule, Y ishydrogen or a metal or together with X defines the covalent bond, eachoccurance of R₁ and R₃ is independently any nucleic acid residue, m isan integer between 1 and 3000 or zero , n is an integer between 1 and3000 or zero, and R₂ is a nucleic acid sequence of the invention,particularly a nucleic acid sequence selected from Table 1. In thepolynucleotide formula above R₂ is oriented so that its 5' end residueis at the left, bound to R₁ and its 3' end residue is at the right,bound to R₃. Any stretch of nucleic acid residues denoted by either Rgroup, where m and/or n is greater than 1, may be either a heteropolymeror a homopolymer, preferably a heteropolymer. Where, in a preferredembodiment, X and Y together define a covalent bond, the polynucleotideof the above formula is a closed, circular polynucleotide, which can bea double-stranded polynucleotide wherein the formula shows a firststrand to which the second strand is complementary. In another preferredembodiment m and/or n is an integer between 1 and 1000.

It is most preferred that the polynucleotides of the inventions arederived from Staphylococcus aureus, however, they may preferably beobtained from organisms of the same taxonomic genus. They may also beobtained, for example, from organisms of the same taxonomic family ororder.

The term "polynucleotide encoding a polypeptide" as used hereinencompasses polynucleotides that include a sequence encoding apolypeptide of the invention, particularly a bacterial polypeptide andmore particularly a polypeptide of the Staphylococcus aureus mecB havingan amino acid sequence set out in Table 1 [SEQ ID NO:2 or 4]. The termalso encompasses polynucleotides that include a single continuous regionor discontinuous regions encoding the polypeptide (for example,interrupted by integrated phage or an insertion sequence or editing)together with additional regions, that also may contain coding and/ornon-coding sequences.

The invention further relates to variants of the polynucleotidesdescribed herein that encode for variants of the polypeptide having adeduced amino acid sequence of Table 1 [SEQ ID NO:2 or 4]. Variants thatare fragments of the polynucleotides of the invention may be used tosynthesize full-length polynucleotides of the invention.

Further particularly preferred embodiments are polynucleotides encodingmecB variants, that have the amino acid sequence of mecB polypeptide ofTable 1 [SEQ ID NO:2 or 4] in which several, a few, 5 to 10, 1 to 5, 1to 3, 2, 1 or no amino acid residues are substituted, deleted or added,in any combination. Especially preferred among these are silentsubstitutions, additions and deletions, that do not alter the propertiesand activities of mecB.

Further preferred embodiments of the invention are polynucleotides thatare at least 70% identical over their entire length to a polynucleotideencoding mecB polypeptide having an amino acid sequence set out in Table1 [SEQ ID NO:2 or 4], and polynucleotides that are complementary to suchpolynucleotides. Alternatively, most highly preferred arepolynucleotides that comprise a region that is at least 80% identicalover its entire length to a polynucleotide encoding mecB polypeptide andpolynucleotides complementary thereto. In this regard, polynucleotidesat least 90% identical over their entire length to the same areparticularly preferred, and among these particularly preferredpolynucleotides, those with at least 95% are especially preferred.Furthermore, those with at least 97% are highly preferred among thosewith at least 95%, and among these those with at least 98% and at least99% are particularly highly preferred, with at least 99% being the morepreferred.

Preferred embodiments are polynucleotides that encode polypeptides thatretain substantially the same biological function or activity as themature polypeptide encoded by a DNA of Table 1 [SEQ ID NO: 1 or 3].

The invention further relates to polynucleotides that hybridize to theherein above-described sequences. In this regard, the inventionespecially relates to polynucleotides that hybridize under stringentconditions to the herein above-described polynucleotides. As hereinused, the terms "stringent conditions" and "stringent hybridizationconditions" mean hybridization will occur only if there is at least 95%and preferably at least 97% identity between the sequences. An exampleof stringent hybridization conditions is overnight incubation at 42° C.in a solution comprising: 50% formamide, 5× SSC (150 mM NaCl, 15 mMtrisodium citrate), 50 mM sodium phosphate (pH7.6), 5× Denhardt'ssolution, 10% dextran sulfate, and 20 micrograms/ml denatured, shearedsalmon sperm DNA, followed by washing the hybridization support in0.1×SSC at about 65° C. Hybridization and wash conditions are well knownand exemplified in Sambrook, et al., Molecular Cloning: A LaboratoryManual, Second Edition, Cold Spring Harbor, N.Y., (1989), particularlyChapter 11 therein.

The invention also provides a polynucleotide consisting essentially of apolynucleotide sequence obtainable by screening an appropriate librarycontaining the complete gene for a polynucleotide sequence set forth inSEQ ID NO:1 under stringent hybridization conditions with a probe havingthe sequence of said polynucleotide sequence set forth in SEQ ID NO:1 ora fragment thereof; and isolating said DNA sequence. Fragments usefulfor obtaining such a polynucleotide include, for example, probes andprimers described elsewhere herein.

As discussed additionally herein regarding polynucleotide assays of theinvention, for instance, polynucleotides of the invention as discussedabove, may be used as a hybridization probe for RNA, cDNA and genomicDNA to isolate full-length cDNAs and genomic clones encoding mecB and toisolate cDNA and genomic clones of other genes that have a high sequencesimilarity to the mecB gene. Such probes generally will comprise atleast 15 bases. Preferably, such probes will have at least 30 bases andmay have at least 50 bases. Particularly preferred probes will have atleast 30 bases and will have 50 bases or less.

For example, the coding region of the mecB gene may be isolated byscreening using a DNA sequence provided in Table 1 [SEQ ID NO: 1 or 3]to synthesize an oligonucleotide probe. A labeled oligonucleotide havinga sequence complementary to that of a gene of the invention is then usedto screen a library of cDNA, genomic DNA or mRNA to determine whichmembers of the library the probe hybridizes to.

The polynucleotides and polypeptides of the invention may be employed,for example, as research reagents and materials for discovery oftreatments of and diagnostics for disease, particularly human disease,as further discussed herein relating to polynucleotide assays.

Polynucleotides of the invention that are oligonucleotides derived fromthe sequences of Table 1 [SEQ ID NOS: 1 or 2 or 3 or 4] may be used inthe processes herein as described, but preferably for PCR, to determinewhether or not the polynucleotides identified herein in whole or in partare transcribed in bacteria in infected tissue. It is recognized thatsuch sequences will also have utility in diagnosis of the stage ofinfection and type of infection the pathogen has attained.

The invention also provides polynucleotides that may encode apolypeptide that is the mature protein plus additional amino orcarboxyl-terminal amino acids, or amino acids interior to the maturepolypeptide (when the mature form has more than one polypeptide chain,for instance). Such sequences may play a role in processing of a proteinfrom precursor to a mature form, may allow protein transport, maylengthen or shorten protein half-life or may facilitate manipulation ofa protein for assay or production, among other things. As generally isthe case in vivo, the additional amino acids may be processed away fromthe mature protein by cellular enzymes.

A precursor protein, having the mature form of the polypeptide fused toone or more prosequences may be an inactive form of the polypeptide.When prosequences are removed such inactive precursors generally areactivated. Some or all of the prosequences may be removed beforeactivation. Generally, such precursors are called proproteins.

In addition to the standard A, G, C, T/U representations for nucleicacid bases, the term "N" may also be used in describing certainpolynucleotides of the invention. "N" means that any of the four DNA orRNA bases may appear at such a designated position in the DNA or RNAsequence, except it is preferred that N is not a base that when taken incombination with adjacent nucleotide positions, when read in the correctreading frame, would have the effect of generating a prematuretermination codon in such reading frame.

In sum, a polynucleotide of the invention may encode a mature protein, amature protein plus a leader sequence (which may be referred to as apreprotein), a precursor of a mature protein having one or moreprosequences that are not the leader sequences of a preprotein, or apreproprotein, which is a precursor to a proprotein, having a leadersequence and one or more prosequences, which generally are removedduring processing steps that produce active and mature forms of thepolypeptide.

Vectors, Host Cells, Expression

The invention also relates to vectors that comprise a polynucleotide orpolynucleotides of the invention, host cells that are geneticallyengineered with vectors of the invention and the production ofpolypeptides of the invention by recombinant techniques. Cell-freetranslation systems can also be employed to produce such proteins usingRNAs derived from the DNA constructs of the invention.

For recombinant production, host cells can be genetically engineered toincorporate expression systems or portions thereof or polynucleotides ofthe invention. Introduction of a polynucleotide into the host cell canbe effected by methods described in many standard laboratory manuals,such as Davis et al., BASIC METHODS IN MOLECULAR BIOLOGY, (1986) andSambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed., ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989), suchas, calcium phosphate transfection, DEAE-dextran mediated transfection,transvection, microinjection, cationic lipid-mediated transfection,electroporation, transduction, scrape loading, ballistic introductionand infection.

Representative examples of appropriate hosts include bacterial cells,such as streptococci, staphylococci, enterococci E. coli, streptomycesand Bacillus subtilis cells; fungal cells, such as yeast cells andAspergillus cells; insect cells such as Drosophila S2 and Spodoptera Sf9cells; animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, 293 andBowes melanoma cells; and plant cells.

A great variety of expression systems can be used to produce thepolypeptides of the invention. Such vectors include, among others,chromosomal, episomal and virus-derived vectors, e.g., vectors derivedfrom bacterial plasmids, from bacteriophage, from transposons, fromyeast episomes, from insertion elements, from yeast chromosomalelements, from viruses such as baculoviruses, papova viruses, such asSV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabiesviruses and retroviruses, and vectors derived from combinations thereof,such as those derived from plasmid and bacteriophage genetic elements,such as cosmids and phagemids. The expression system constructs maycontain control regions that regulate as well as engender expression.Generally, any system or vector suitable to maintain, propagate orexpress polynucleotides and/or to express a polypeptide in a host may beused for expression in this regard. The appropriate DNA sequence may beinserted into the expression system by any of a variety of well-knownand routine techniques, such as, for example, those set forth inSambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, (supra).

For secretion of the translated protein into the lumen of theendoplasmic reticulum, into the periplasmic space or into theextracellular environment, appropriate secretion signals may beincorporated into the expressed polypeptide. These signals may beendogenous to the polypeptide or they may be heterologous signals.

Polypeptides of the invention can be recovered and purified fromrecombinant cell cultures by well-known methods including ammoniumsulfate or ethanol precipitation, acid extraction, anion or cationexchange chromatography, phosphocellulose chromatography, hydrophobicinteraction chromatography, affinity chromatography, hydroxylapatitechromatography, and lectin chromatography. Most preferably, highperformance liquid chromatography is employed for purification. Wellknown techniques for refolding protein may be employed to regenerateactive conformation when the polypeptide is denatured during isolationand or purification.

Diagnostic Assays

This invention is also related to the use of the mecB polynucleotides ofthe invention for use as diagnostic reagents. Detection of mecB in aeukaryote, particularly a mammal, and especially a human, will provide adiagnostic method for diagnosis of a disease. Eukaryotes (herein also"individual(s)"), particularly mammals, and especially humans,particularly those infected or suspected to be infected with an organismcomprising the mecB gene may be detected at the nucleic acid level by avariety of techniques.

Nucleic acids for diagnosis may be obtained from an infectedindividual's cells and tissues, such as bone, blood, muscle, cartilage,and skin. Genomic DNA may be used directly for detection or may beamplified enzymatically by using PCR or other amplification techniqueprior to analysis. RNA, cDNA and genomic DNA may also be used in thesame ways. Using amplification, characterization of the species andstrain of prokaryote present in an individual, may be made by ananalysis of the genotype of the prokaryote gene. Deletions andinsertions can be detected by a change in size of the amplified productin comparison to the genotype of a reference sequence. Point mutationscan be identified by hybridizing amplified DNA to labeled mecBpolynucleotide sequences. Perfectly matched sequences can bedistinguished from mismatched duplexes by RNase digestion or bydifferences in melting temperatures. DNA sequence differences may alsobe detected by alterations in the electrophoretic mobility of the DNAfragments in gels, with or without denaturing agents, or by direct DNAsequencing. See, e.g., Myers et al., Science, 230: 1242 (1985). Sequencechanges at specific locations also may be revealed by nucleaseprotection assays, such as RNase and S1 protection or a chemicalcleavage method. See, e.g., Cotton et al., Proc. Natl. Acad. Sci., USA,85: 4397-4401 (1985).

Cells carrying mutations or polymorphisms in the gene of the inventionmay also be detected at the DNA level by a variety of techniques, toallow for serotyping, for example. For example, RT-PCR can be used todetect mutations. It is particularly preferred to used RT-PCR inconjunction with automated detection systems, such as, for example,GeneScan. RNA, cDNA or genomic DNA may also be used for the samepurpose, PCR or RT-PCR. As an example, PCR primers complementary to anucleic acid encoding mecB can be used to identify and analyzemutations. Examples of representative primers are shown below in Table2.

                  TABLE 2                                                         ______________________________________                                        Primers for amplification of mecB polynucleotides                             SEQ ID NO  PRIMER SEQUENCE                                                    ______________________________________                                        5          5'-atagcaactgtatctactactgaagg-3'                                   6                         5'-aattgaagtattaagtagacgtacg-3'                     ______________________________________                                    

The invention also includes primers of the formula:

    X--(R.sub.1).sub.m --(R.sub.2)--(R.sub.3).sub.n --Y

wherein, at the 5' end of the molecule, X is hydrogen, and at the 3' endof the molecule, Y is hydrogen or a metal, R₁ and R₃ is any nucleic acidresidue, m is an integer between 1 and 20 or zero, n is an integerbetween 1 and 20 or zero, and R₂ is a primer sequence of the invention,particularly a primer sequence selected from Table 2. In thepolynucleotide formula above R₂ is oriented so that its 5' end residueis at the left, bound to R₁, and its 3' end residue is at the right,bound to R₃. Any stretch of nucleic acid residues denoted by either Rgroup, where m and/or n is greater than 1, may be either a heteropolymeror a homopolymer, preferably a heteropolymer being complementary to aregion of a polynucleotide of Table 1. In a preferred embodiment mand/or n is an integer between 1 and 10.

The invention further provides these primers with 1, 2, 3 or 4nucleotides removed from the 5' and/or the 3' end. These primers may beused for, among other things, amplifying mecB DNA isolated from a samplederived from an individual. The primers may be used to amplify the geneisolated from an infected individual such that the gene may then besubject to various techniques for elucidation of the DNA sequence. Inthis way, mutations in the DNA sequence may be detected and used todiagnose infection and to serotype and/or classify the infectious agent.

The invention further provides a process for diagnosing, disease,preferably bacterial infections, more preferably infections byStaphylococcus aureus, comprising determining from a sample derived froman individual a increased level of expression of polynucleotide having asequence of Table 1 [SEQ ID NO: 1 or 3]. Increased or decreasedexpression of mecB polynucleotide can be measured using any on of themethods well known in the art for the quantation of polynucleotides,such as, for example, amplification, PCR, RT-PCR, RNase protection,Northern blotting and other hybridization methods.

In addition, a diagnostic assay in accordance with the invention fordetecting over-expression of mecB protein compared to normal controltissue samples may be used to detect the presence of an infection, forexample. Assay techniques that can be used to determine levels of a mecBprotein, in a sample derived from a host are well-known to those ofskill in the art. Such assay methods include radioimmunoassays,competitive-binding assays, Western Blot analysis and ELISA assays.

Antibodies

The polypeptides of the invention or variants thereof, or cellsexpressing them can be used as an immunogen to produce antibodiesimmunospecific for such polypeptides. "Antibodies" as used hereinincludes monoclonal and polyclonal antibodies, chimeric, single chain,simianized antibodies and humanized antibodies, as well as Fabfragments, including the products of an Fab immunolglobulin expressionlibrary.

Antibodies generated against the polypeptides of the invention can beobtained by administering the polypeptides or epitope-bearing fragments,analogues or cells to an animal, preferably a nonhuman, using routineprotocols. For preparation of monoclonal antibodies, any technique knownin the art that provides antibodies produced by continuous cell linecultures can be used. Examples include various techniques, such as thosein Kohler, G. and Milstein, C., Nature 256: 495-497 (1975); Kozbor etal., Immunology Today 4: 72 (1983); Cole et al., pg. 77-96 in MONOCLONALANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc. (1985).

Techniques for the production of single chain antibodies (U.S. Pat. No.4,946,778) can be adapted to produce single chain antibodies topolypeptides of this invention. Also, transgenic mice, or otherorganisms such as other mammals, may be used to express humanizedantibodies.

Alternatively phage display technology may be utilized to selectantibody genes with binding activities towards the polypeptide eitherfrom repertoires of PCR amplified v-genes of lymphocytes from humansscreened for possessing anti-mecB or from naive libraries (McCafferty,J. et al., (1990), Nature 348, 552-554; Marks, J. et al., (1992)Biotechnology 10, 779-783). The affinity of these antibodies can also beimproved by chain shuffling (Clackson, T. et al., (1991) Nature 352,624-628).

If two antigen binding domains are present each domain may be directedagainst a different epitope--termed `bispecific` antibodies.

The above-described antibodies may be employed to isolate or to identifyclones expressing the polypeptides to purify the polypeptides byaffinity chromatography.

Thus, among others, antibodies against mecB-polypeptide may be employedto treat infections, particularly bacterial infections.

Polypeptide variants include antigenically, epitopically orimmunologically equivalent variants that form a particular aspect ofthis invention. The term "antigenically equivalent derivative" as usedherein encompasses a polypeptide or its equivalent which will bespecifically recognized by certain antibodies which, when raised to theprotein or polypeptide according to the invention, interfere with theimmediate physical interaction between pathogen and mammalian host. Theterm "immunologically equivalent derivative" as used herein encompassesa peptide or its equivalent which when used in a suitable formulation toraise antibodies in a vertebrate, the antibodies act to interfere withthe immediate physical interaction between pathogen and mammalian host.

The polypeptide, such as an antigenically or immunologically equivalentderivative or a fusion protein thereof is used as an antigen to immunizea mouse or other animal such as a rat or chicken. The fusion protein mayprovide stability to the polypeptide. The antigen may be associated, forexample by conjugation, with an immunogenic carrier protein for examplebovine serum albumin (BSA) or keyhole limpet haemocyanin (KLH).Alternatively a multiple antigenic peptide comprising multiple copies ofthe protein or polypeptide, or an antigenically or immunologicallyequivalent polypeptide thereof may be sufficiently antigenic to improveimmunogenicity so as to obviate the use of a carrier.

Preferably, the antibody or variant thereof is modified to make it lessimmunogenic in the individual. For example, if the individual is humanthe antibody may most preferably be "humanized"; where thecomplimentarity determining region(s) of the hybridoma-derived antibodyhas been transplanted into a human monoclonal antibody, for example asdescribed in Jones, P. et al. (1986), Nature 321, 522-525 or Tempest etal., (1991) Biotechnology 9, 266-273.

The use of a polynucleotide of the invention in genetic immunizationwill preferably employ a suitable delivery method such as directinjection of plasmid DNA into muscles (Wolff et al., Hum Mol Genet 1992,1:363, Manthorpe et al., Hum. Gene Ther. 1963:4, 419), delivery of DNAcomplexed with specific protein carriers (Wu et al., J Biol Chem. 1989:264,16985), coprecipitation of DNA with calcium phosphate (3envenisty &Reshef, PNAS USA, 1986:83,9551), encapsulation of DNA in various formsof liposomes (Kaneda et al., Science 1989:243,375), particle bombardment(Tang et al., Nature 1992, 356:152, Eisenbraun et al., DNA Cell Biol1993, 12:791) and in vivo infection using cloned retroviral vectors(Seeger et al., PNAS USA 1984:81,5849).

Antagonists and Agonists--Assays and Molecules

Polypeptides of the invention may also be used to assess the binding ofsmall molecule substrates and ligands in, for example, cells, cell-freepreparations, chemical libraries, and natural product mixtures. Thesesubstrates and ligands may be natural substrates and ligands or may bestructural or functional mimetics. See, e.g., Coligan et al., CurrentProtocols in Immunology 1(2): Chapter 5 (1991).

The invention also provides a method of screening compounds to identifythose which enhance (agonist) or block (antagonist) the action of mecBpolypeptides or polynucleotides, particularly those compounds that arebacteriostatic and/or bacteriocidal. The method of screening may involvehigh-throughput techniques. For example, to screen for agonists orantagoists, a synthetic reaction mix, a cellular compartment, such as amembrane, cell envelope or cell wall, or a preparation of any thereof,comprising mecB polypeptide and a labeled substrate or ligand of suchpolypeptide is incubated in the absence or the presence of a candidatemolecule that may be a mecB agonist or antagonist. The ability of thecandidate molecule to agonize or antagonize the mecB polypeptide isreflected in decreased binding of the labeled ligand or decreasedproduction of product from such substrate. Molecules that bindgratuitously, i.e., without inducing the effects of mecB polypeptide aremost likely to be good antagonists. Molecules that bind well andincrease the rate of product production from substrate are agonists.Detection of the rate or level of production of product from substratemay be enhanced by using a reporter system. Reporter systems that may beuseful in this regard include but are not limited to colorimetriclabeled substrate converted into product, a reporter gene that isresponsive to changes in mecB polynucleotide or polypeptide activity,and binding assays known in the art.

Another example of an assay for mecB antagonists is a competitive assaythat combines mecB and a potential antagonist with mecB-bindingmolecules, recombinant mecB binding molecules, natural substrates orligands, or substrate or ligand mimetics, under appropriate conditionsfor a competitive inhibition assay. MecB can be labeled, such as byradioactivity or a colorimetric compound, such that the number of mecBmolecules bound to a binding molecule or converted to product can bedetermined accurately to assess the effectiveness of the potentialantagonist.

Potential antagonists include small organic molecules, peptides,polypeptides and antibodies that bind to a polynucleotide or polypeptideof the invention and thereby inhibit or extinguish its activity.Potential antagonists also may be small organic molecules, a peptide, apolypeptide such as a closely related protein or antibody that binds thesame sites on a binding molecule, such as a binding molecule, withoutinducing mecB-induced activities, thereby preventing the action of mecBby excluding mecB from binding.

Potential antagonists include a small molecule that binds to andoccupies the binding site of the polypeptide thereby preventing bindingto cellular binding molecules, such that normal biological activity isprevented. Examples of small molecules include but are not limited tosmall organic molecules, peptides or peptide-like molecules. Otherpotential antagonists include antisense molecules (see Okano, J.Neurochem. 56: 560 (1991); OLIGODEOXYNUCLEOTIDES AS ANTISENSE INHIBITORSOF GENE EXPRESSION, CRC Press, Boca Raton, Fla. (1988), for adescription of these molecules). Preferred potential antagonists includecompounds related to and variants of mecB.

Each of the DNA sequences provided herein may be used in the discoveryand development of antibacterial compounds. The encoded protein, uponexpression, can be used as a target for the screening of antibacterialdrugs. Additionally, the DNA sequences encoding the amino terminalregions of the encoded protein or Shine-Delgamo or other translationfacilitating sequences of the respective mRNA can be used to constructantisense sequences to control the expression of the coding sequence ofinterest.

The invention also provides the use of the polypeptide, polynucleotideor inhibitor of the invention to interfere with the initial physicalinteraction between a pathogen and mammalian host responsible forsequelae of infection. In particular the molecules of the invention maybe used: in the prevention of adhesion of bacteria, in particular grampositive bacteria, to mammalian extracellular matrix proteins onin-dwelling devices or to extracellular matrix proteins in wounds; toblock mecB protein-mediated mammalian cell invasion by, for example,initiating phosphorylation of mammalian tyrosine kinases (Rosenshine etal., Infect. Immun. 60:2211 (1992); to block bacterial adhesion betweenmammalian extracellular matrix proteins and bacterial mecB proteins thatmediate tissue damage and; to block the normal progression ofpathogenesis in infections initiated other than by the implantation ofin-dwelling devices or by other surgical techniques.

The antagonists and agonists of the invention may be employed, forinstance, to inhibit and treat diseases.

Helicobacter pylori (herein H. pylori) bacteria infect the stomachs ofover one-third of the world's population causing stomach cancer, ulcers,and gastritis (International Agency for Research on Cancer (1994)Schistosomes, Liver Flukes and Helicobacter Pylori (International Agencyfor Research on Cancer, Lyon, France;http://www.uicc.ch/ecp/ecp2904.htm). Moreover, the international Agencyfor Research on Cancer recently recognized a cause-and-effectrelationship between H. pylori and gastric adenocarcinoma, classifyingthe bacterium as a Group I (definite) carcinogen. Preferredantimicrobial compounds of the invention (agonists and antagonists ofmecB) found using screens provided by the invention, particularlybroad-spectrum antibiotics, should be useful in the treatment of H.pylori infection. Such treatment should decrease the advent of H.pylori-induced cancers, such as gastrointestinal carcinoma.

Such treatment should also cure gastric ulcers and gastritis.

Vaccines

Another aspect of the invention relates to a method for inducing animmunological response in an individual, particularly a mammal whichcomprises inoculating the individual with mecB, or a fragment or variantthereof, adequate to produce antibody and/or T cell immune response toprotect said individual from infection, particularly bacterial infectionand most particularly Staphylococcus aureus infection. Also provided aremethods whereby such immunological response slows bacterial replication.Yet another aspect of the invention relates to a method of inducingimmunological response in an individual which comprises delivering tosuch individual a nucleic acid vector to direct expression of mecB, or afragment or a variant thereof, for expressing mecB, or a fragment or avariant thereof in vivo in order to induce an immunological response,such as, to produce antibody and/or T cell immune response, including,for example, cytokine-producing T cells or cytotoxic T cells, to protectsaid individual from disease, whether that disease is alreadyestablished within the individual or not. One way of administering thegene is by accelerating it into the desired cells as a coating onparticles or otherwise. Such nucleic acid vector may comprise DNA, RNA,a modified nucleic acid, or a DNA/RNA hybrid.

A further aspect of the invention relates to an immunologicalcomposition which, when introduced into an individual capable or havinginduced within it an immunological response, induces an immunologicalresponse in such individual to a mecB or protein coded therefrom,wherein the composition comprises a recombinant mecB or protein codedtherefrom comprising DNA which codes for and expresses an antigen ofsaid mecB or protein coded therefrom. The immunological response may beused therapeutically or prophylactically and may take the form ofantibody immunity or cellular immunity such as that arising from CTL orCD4+ T cells.

A mecB polypeptide or a fragment thereof may be fused with co-proteinwhich may not by itself produce antibodies, but is capable ofstabilizing the first protein and producing a fused protein which willhave immunogenic and protective properties. Thus fused recombinantprotein, preferably further comprises an antigenic co-protein, such aslipoprotein D from Hemophilus influenzae, Glutathione-S-transferase(GST) or beta-galactosidase, relatively large co-proteins whichsolubilize the protein and facilitate production and purificationthereof. Moreover, the co-protein may act as an adjuvant in the sense ofproviding a generalized stimulation of the immune system. The co-proteinmay be attached to either the amino or carboxy terminus of the firstprotein.

Provided by this invention are compositions, particularly vaccinecompositions, and methods comprising the polypeptides or polynucleotidesof the invention and immunostimulatory DNA sequences, such as thosedescribed in Sato, Y. et al. Science 273: 352 (1996).

Also, provided by this invention are methods using the describedpolynucleotide or particular fragments thereof which have been shown toencode non-variable regions of bacterial cell surface proteins in DNAconstructs used in such genetic immunization experiments in animalmodels of infection with Staphylococcus aureus will be particularlyuseful for identifying protein epitopes able to provoke a prophylacticor therapeutic immune response. It is believed that this approach willallow for the subsequent preparation of monoclonal antibodies ofparticular value from the requisite organ of the animal successfullyresisting or clearing infection for the development of prophylacticagents or therapeutic treatments of bacterial infection, particularlyStaphylococcus aureus infection, in mammals, particularly humans.

The polypeptide may be used as an antigen for vaccination of a host toproduce specific antibodies which protect against invasion of bacteria,for example by blocking adherence of bacteria to damaged tissue.Examples of tissue damage include wounds in skin or connective tissuecaused, e.g., by mechanical, chemical or thermal damage or byimplantation of indwelling devices, or wounds in the mucous membranes,such as the mouth, mammary glands, urethra or vagina.

The invention also includes a vaccine formulation which comprises animmunogenic recombinant protein of the invention together with asuitable carrier. Since the protein may be broken down in the stomach,it is preferably administered parenterally, including, for example,administration that is subcutaneous, intramuscular, intravenous, orintradermal. Formulations suitable for parenteral administration includeaqueous and non-aqueous sterile injection solutions which may containanti-oxidants, buffers, bacteriostats and solutes which render theformulation insotonic with the bodily fluid, preferably the blood, ofthe individual; and aqueous and non-aqueous sterile suspensions whichmay include suspending agents or thickening agents. The formulations maybe presented in unit-dose or multi-dose containers, for example, sealedampules and vials and may be stored in a freeze-dried conditionrequiring only the addition of the sterile liquid carrier immediatelyprior to use. The vaccine formulation may also include adjuvant systemsfor enhancing the immunogenicity of the formulation, such as oil-inwater systems and other systems known in the art. The dosage will dependon the specific activity of the vaccine and can be readily determined byroutine experimentation.

While the invention has been described with reference to certain mecBprotein, it is to be understood that this covers fragments of thenaturally occurring protein and similar proteins with additions,deletions or substitutions which do not substantially affect theimmunogenic properties of the recombinant protein.

Compositions, Kits and Administration

The invention also relates to compositions comprising the polynucleotideor the polypeptides discussed above or their agonists or antagonists.The polypeptides of the invention may be employed in combination with anon-sterile or sterile carrier or carriers for use with cells, tissuesor organisms, such as a pharmaceutical carrier suitable foradministration to a subject. Such compositions comprise, for instance, amedia additive or a therapeutically effective amount of a polypeptide ofthe invention and a pharmaceutically acceptable carrier or excipient.Such carriers may include, but are not limited to, saline, bufferedsaline, dextrose, water, glycerol, ethanol and combinations thereof. Theformulation should suit the mode of administration. The inventionfurther relates to diagnostic and pharmaceutical packs and kitscomprising one or more containers filled with one or more of theingredients of the aforementioned compositions of the invention.

Polypeptides and other compounds of the invention may be employed aloneor in conjunction with other compounds, such as therapeutic compounds.

The pharmaceutical compositions may be administered in any effective,convenient manner including, for instance, administration by topical,oral, anal, vaginal, intravenous, intraperitoneal, intramuscular,subcutaneous, intranasal or intradermal routes among others.

In therapy or as a prophylactic, the active agent may be administered toan individual as an injectable composition, for example as a sterileaqueous dispersion, preferably isotonic.

Alternatively the composition may be formulated for topical applicationfor example in the form of ointments, creams, lotions, eye ointments,eye drops, ear drops, mouthwash, impregnated dressings and sutures andaerosols, and may contain appropriate conventional additives, including,for example, preservatives, solvents to assist drug penetration, andemollients in ointments and creams. Such topical formulations may alsocontain compatible conventional carriers, for example cream or ointmentbases, and ethanol or oleyl alcohol for lotions. Such carriers mayconstitute from about 1% to about 98% by weight of the formulation; moreusually they will constitute up to about 80% by weight of theformulation.

For administration to mammals, and particularly humans, it is expectedthat the daily dosage level of the active agent will be from 0.01 mg/kgto 10 mg/kg, typically around 1 mg/kg. The physician in any event willdetermine the actual dosage which will be most suitable for anindividual and will vary with the age, weight and response of theparticular individual. The above dosages are exemplary of the averagecase. There can, of course, be individual instances where higher orlower dosage ranges are merited, and such are within the scope of thisinvention.

In-dwelling devices include surgical implants, prosthetic devices andcatheters, i.e., devices that are introduced to the body of anindividual and remain in position for an extended time. Such devicesinclude, for example, artificial joints, heart valves, pacemakers,vascular grafts, vascular catheters, cerebrospinal fluid shunts, urinarycatheters, continuous ambulatory peritoneal dialysis (CAPD) catheters.

The composition of the invention may be administered by injection toachieve a systemic effect against relevant bacteria shortly beforeinsertion of an in-dwelling device. Treatment may be continued aftersurgery during the in-body time of the device. In addition, thecomposition could also be used to broaden perioperative cover for anysurgical technique to prevent bacterial wound infections, especiallyStaphylococcus aureus wound infections.

Many orthopaedic surgeons consider that humans with prosthetic jointsshould be considered for antibiotic prophylaxis before dental treatmentthat could produce a bacteremia. Late deep infection is a seriouscomplication sometimes leading to loss of the prosthetic joint and isaccompanied by significant morbidity and mortality. It may therefore bepossible to extend the use of the active agent as a replacement forprophylactic antibiotics in this situation.

In addition to the therapy described above, the compositions of thisinvention may be used generally as a wound treatment agent to preventadhesion of bacteria to matrix proteins exposed in wound tissue and forprophylactic use in dental treatment as an alternative to, or inconjunction with, antibiotic prophylaxis.

Alternatively, the composition of the invention may be used to bathe anindwelling device immediately before insertion. The active agent willpreferably be present at a concentration of 1 μg/ml to 10 mg/ml forbathing of wounds or indwelling devices.

A vaccine composition is conveniently in injectable form. Conventionaladjuvants may be employed to enhance the immune response. A suitableunit dose for vaccination is 0.5-5 microgram/kg of antigen, and suchdose is preferably administered 1-3 times and with an interval of 1-3weeks. With the indicated dose range, no adverse toxicological effectswill be observed with the compounds of the invention which wouldpreclude their administration to suitable individuals.

Each reference disclosed herein is incorporated by reference herein inits entirety. Any patent application to which this application claimspriority is also incorporated by reference herein in its entirety.

GLOSSARY

The following definitions are provided to facilitate understanding ofcertain terms used frequently herein.

"Disease(s)" means and disease caused by or related to infection by abacteria, including disease, such as, infections of the upperrespiratory tract (e.g., otitis media, bacterial tracheitis, acuteepiglottitis, thyroiditis), lower respiratory (e.g., empyema, lungabscess), cardiac (e.g., infective endocarditis), gastrointestinal(e.g., secretory diarrhoea, splenic absces, retroperitoneal abscess),CNS (e.g., cerebral abscess), eye (e.g., blepharitis, conjunctivitis,keratitis, endophthalmitis, preseptal and orbital cellultis,darcryocystitis), kidney and urinary tract (e.g., epididymitis,intrarenal and perinephric absces, toxic shock syndrome), skin (e.g.,impetigo, folliculitis, cutaneous abscesses, cellulitis, woundinfection, bacterial myositis) bone and joint (e.g., septic arthritis,osteomyelitis).

"Host cell" is a cell which has been transformed or transfected, or iscapable of transformation or transfection by an exogenous polynucleotidesequence.

"Identity," as known in the art, is a relationship between two or morepolypeptide sequences or two or more polynucleotide sequences, as thecase may be, as determined by comparing the sequences. In the art,"identity" also means the degree of sequence relatedness betweenpolypeptide or polynucleotide sequences, as the case may be, asdetermined by the match between strings of such sequences. "Identity"can be readily calculated by known methods, including but not limited tothose described in (Computational Molecular Biology, Lesk, A. M., ed.,Oxford University Press, New York, 1988; Biocomputing: Informatics andGenome Projects, Smith, D. W., ed., Academic Press, New York, 1993;Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin,H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis inMolecular Biology, von Heinje, G., Academic Press, 1987; and SequenceAnalysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press,New York, 1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math.,48: 1073 (1988). Methods to determine identity are designed to give thelargest match between the sequences tested. Moreover, methods todetermine identity are codified in publicly available computer programs.Computer program methods to determine identity between two sequencesinclude, but are not limited to, the GCG program package (Devereux, J.,et al., Nucleic Acids Research 12(1): 387 (1984)), BLASTP, BLASTN, andFASTA (Atschul, S. F. et al., J. Molec. Biol. 215: 403-410 (1990). TheBLAST X program is publicly available from NCBI and other sources (BLASTManual, Altschul, S., et al., NCBI NLM NIH Bethesda, Md. 20894;Altschul, S., et al., J. Mol. Biol. 215: 403-410 (1990). The well knownSmith Waterman algorithm may also be used to determine identity.

Parameters for polypeptide sequence comparison include the following:

1) Algorithm: Needleman and Wunsch, J. Mol Biol. 48: 443-453 (1970)

Comparison matrix: BLOSSUM62 from Hentikoff and Hentikoff, Proc. Natl.Acad. Sci. USA. 89:10915-10919 (1992)

Gap Penalty: 12

Gap Length Penalty: 4

A program useful with these parameters is publicly available as the"gap" program from Genetics Computer Group, Madison Wis. Theaforementioned parameters are the default parameters for peptidecomparisons (along with no penalty for end gaps).

Parameters for polynucleotide comparison include the following:

1) Algorithm: Needleman and Wunsch, J. Mol Biol. 48: 443-453 (1970)

Comparison matrix: matches=+10, mismatch=0

Gap Penalty: 50

Gap Length Penalty: 3

Available as: The "gap" program from Genetics Computer Group, MadisonWis. These are the default parameters for nucleic acid comparisons.

A preferred meaning for "identity" for polynucleotides and polypeptides,as the case may be, are provided in (1) and (2) below.

(1) Polynucleotide embodiments further include an isolatedpolynucleotide comprising a polynucleotide sequence having at least a50, 60, 70, 80, 85, 90, 95, 97 or 100% identity to the referencesequence of SEQ ID NO: 1, wherein said polynucleotide sequence may beidentical to the reference sequence of SEQ ID NO: 1 or may include up toa certain integer number of nucleotide alterations as compared to thereference sequence, wherein said alterations are selected from the groupconsisting of at least one nucleotide deletion, substitution, includingtransition and transversion, or insertion, and wherein said alterationsmay occur at the 5' or 3' terminal positions of the reference nucleotidesequence or anywhere between those terminal positions, interspersedeither individually among the nucleotides in the reference sequence orin one or more contiguous groups within the reference sequence, andwherein said number of nucleotide alterations is determined bymultiplying the total number of nucleotides in SEQ ID NO: 1 by theinteger defining the percent identity divided by 100 and thensubtracting that product from said total number of nucleotides in SEQ IDNO: 1, or:

    n.sub.n ≦x.sub.n -(x.sub.n ·y),

wherein n_(n) in is the number of nucleotide alterations, x_(n) is thetotal number of nucleotides in SEQ ID NO:1, y is 0.50 for 50%, 0.60 for60%, 0.70 for 70%, 0.80 for 80%, 0.85 for 85%, 0.90 for 90%, 0.95 for95%, 0.97 for 97% or 1.00 for 100%, and · is the symbol for themultiplication operator, and wherein any non-integer product of x_(n)and y is rounded down to the nearest integer prior to subtracting itfrom x_(n). Alterations of a polynucleotide sequence encoding thepolypeptide of SEQ ID NO:2 may create nonsense, missense or frameshiftmutations in this coding sequence and thereby alter the polypeptideencoded by the polynucleotide following such alterations.

By way of example, a polynucleotide sequence of the present inventionmay be identical to the reference sequence of SEQ ID NO:2, that is itmay be 100% identical, or it may include up to a certain integer numberof amino acid alterations as compared to the reference sequence suchthat the percent identity is less than 100% identity. Such alterationsare selected from the group consisting of at least one nucleic aciddeletion, substitution, including transition and transversion, orinsertion, and wherein said alterations may occur at the 5' or ₃ 'terminal positions of the reference polynucleotide sequence or anywherebetween those terminal positions, interspersed either individually amongthe nucleic acids in the reference sequence or in one or more contiguousgroups within the reference sequence. The number of nucleic acidalterations for a given percent identity is determined by multiplyingthe total number of amino acids in SEQ ID NO:2 by the integer definingthe percent identity divided by 100 and then subtracting that productfrom said total number of amino acids in SEQ ID NO:2, or:

    n.sub.n ≦x.sub.n -(x.sub.n ·y),

wherein n_(n) is the number of amino acid alterations, x_(n) is thetotal number of amino acids in SEQ ID NO:2, y is, for instance 0.70 for70%, 0.80 for 80%, 0.85 for 85% etc., · is the symbol for themultiplication operator, and wherein any non-integer product of x_(n)and y is rounded down to the nearest integer prior to subtracting itfrom x_(n).

(2) Polypeptide embodiments further include an isolated polypeptidecomprising a polypeptide having at least a 50, 60, 70, 80, 85, 90, 95,97 or 100% identity to a polypeptide reference sequence of SEQ ID NO:2,wherein said polypeptide sequence may be identical to the referencesequence of SEQ ID NO: 2 or may include up to a certain integer numberof amino acid alterations as compared to the reference sequence, whereinsaid alterations are selected from the group consisting of at least oneamino acid deletion, substitution, including conservative andnon-conservative substitution, or insertion, and wherein saidalterations may occur at the amino- or carboxy-terminal positions of thereference polypeptide sequence or anywhere between those terminalpositions, interspersed either individually among the amino acids in thereference sequence or in one or more contiguous groups within thereference sequence, and wherein said number of amino acid alterations isdetermined by multiplying the total number of amino acids in SEQ ID NO:2by the integer defining the percent identity divided by 100 and thensubtracting that product from said total number of amino acids in SEQ IDNO:2, or:

    n.sub.a ≦x.sub.a -(x.sub.a ·y),

wherein n_(a) is the number of amino acid alterations, x_(a) is thetotal number of amino acids in SEQ ID NO:2, y is 0.50 for 50%, 0.60 for60%, 0.70 for 70%, 0.80 for 80%, 0.85 for 85%, 0.90 for 90%, 0.95 for95%, 0.97 for 97% or 1.00 for 100%, and · is the symbol for themultiplication operator, and wherein any non-integer product of x_(a)and y is rounded down to the nearest integer prior to subtracting itfrom x_(a).

By way of example, a polypeptide sequence of the present invention maybe identical to the reference sequence of SEQ ID NO:2, that is it may be100% identical, or it may include up to a certain integer number ofamino acid alterations as compared to the reference sequence such thatthe percent identity is less than 100% identity. Such alterations areselected from the group consisting of at least one amino acid deletion,substitution, including conservative and non-conservative substitution,or insertion, and wherein said alterations may occur at the amino- orcarboxy-terminal positions of the reference polypeptide sequence oranywhere between those terminal positions, interspersed eitherindividually among the amino acids in the reference sequence or in oneor more contiguous groups within the reference sequence. The number ofamino acid alterations for a given % identity is determined bymultiplying the total number of amino acids in SEQ ID NO:2 by theinteger defining the percent identity divided by 100 and thensubtracting that product from said total number of amino acids in SEQ IDNO:2, or:

    n.sub.a ≦x.sub.a -(x.sub.a ·y),

wherein n_(a) is the number of amino acid alterations, x_(a) is thetotal number of amino acids in 25 SEQ ID NO:2, y is, for instance 0.70for 70%, 0.80 for 80%, 0.85 for 85% etc., and · is the symbol for themultiplication operator, and wherein any non-integer product of x_(a)and y is rounded down to the nearest integer prior to subtracting itfrom x_(a).

"Isolated" means altered "by the hand of man" from its natural state,i.e., if it occurs in nature, it has been changed or removed from itsoriginal environment, or both. For example, a polynucleotide or apolypeptide naturally present in a living organism is not "isolated,"but the same polynucleotide or polypeptide separated from the coexistingmaterials of its natural state is "isolated", as the term is employedherein. Moreover, a polynucleotide or polypeptide that is introducedinto an organism by transformation, genetic manipulation or by any otherrecombinant method is "isolated" even if it is still present in saidorganism, which organism may be living or non-living.

"Polynucleotide(s)" generally refers to any polyribonucleotide orpolydeoxribonucleotide, which may be unmodified RNA or DNA or modifiedRNA or DNA. "Polynucleotide(s)" include, without limitation, single- anddouble-stranded DNA, DNA that is a mixture of single- anddouble-stranded regions or single-, double- and triple-stranded regions,single- and double-stranded RNA, and RNA that is mixture of single- anddouble-stranded regions, hybrid molecules comprising DNA and RNA thatmay be single-stranded or, more typically, double-stranded, ortriple-stranded regions, or a mixture of single- and double-strandedregions. In addition, "polynucleotide" as used herein refers totriple-stranded regions comprising RNA or DNA or both RNA and DNA. Thestrands in such regions may be from the same molecule or from differentmolecules. The regions may include all of one or more of the molecules,but more typically involve only a region of some of the molecules. Oneof the molecules of a triple-helical region often is an oligonucleotide.As used herein, the term "polynucleotide(s)" also includes DNAs or RNAsas described above that contain one or more modified bases. Thus, DNAsor RNAs with backbones modified for stability or for other reasons are"polynucleotide(s)" as that term is intended herein. Moreover, DNAs orRNAs comprising unusual bases, such as inosine, or modified bases, suchas tritylated bases, to name just two examples, are polynucleotides asthe term is used herein. It will be appreciated that a great variety ofmodifications have been made to DNA and RNA that serve many usefulpurposes known to those of skill in the art. The term"polynucleotide(s)" as it is employed herein embraces such chemically,enzymatically or metabolically modified forms of polynucleotides, aswell as the chemical forms of DNA and RNA characteristic of viruses andcells, including, for example, simple and complex cells."Polynucleotide(s)" also embraces short polynucleotides often referredto as oligonucleotide(s).

"Polypeptide(s)" refers to any peptide or protein comprising two or moreamino acids joined to each other by peptide bonds or modified peptidebonds. "Polypeptide(s)" refers to both short chains, commonly referredto as peptides, oligopeptides and oligomers and to longer chainsgenerally referred to as proteins. Polypeptides may contain amino acidsother than the 20 gene encoded amino acids. "Polypeptide(s)" includethose modified either by natural processes, such as processing and otherpost-translational modifications, but also by chemical modificationtechniques. Such modifications are well described in basic texts and inmore detailed monographs, as well as in a voluminous researchliterature, and they are well known to those of skill in the art. Itwill be appreciated that the same type of modification may be present inthe same or varying degree at several sites in a given polypeptide.Also, a given polypeptide may contain many types of modifications.Modifications can occur anywhere in a polypeptide, including the peptidebackbone, the amino acid side-chains, and the amino or carboxyl termini.Modifications include, for example, acetylation, acylation,ADP-ribosylation, amidation, covalent attachment of flavin, covalentattachment of a heme moiety, covalent attachment of a nucleotide ornucleotide derivative, covalent attachment of a lipid or lipidderivative, covalent attachment of phosphotidylinositol, cross-linking,cyclization, disulfide bond formation, demethylation, formation ofcovalent cross-links, formation of cysteine, formation of pyroglutamate,formylation, gamma-carboxylation, glycosylation, GPI anchor formation,hydroxylation, iodination, methylation, myristoylation, oxidation,proteolytic processing, phosphorylation, prenylation, racemization,glycosylation, lipid attachment, sulfation, gamma-carboxylation ofglutamic acid residues, hydroxylation and ADP-ribosylation,selenoylation, sulfation, transfer-RNA mediated addition of amino acidsto proteins, such as arginylation, and ubiquitination. See, forinstance, PROTEINS--STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed., T. E.Creighton, W. H. Freeman and Company, New York (1993) and Wold, F.,Posttranslational Protein Modifications: Perspectives and Prospects,pgs. 1-12 in POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C.Johnson, Ed., Academic Press, New York (1983); Seifter et al., Meth.Enzymol. 182:626-646 (1990) and Rattan et al., Protein Synthesis:Posttranslational Modifications and Aging, Ann. N.Y. Acad. Sci. 663:48-62 (1992). Polypeptides may be branched or cyclic, with or withoutbranching. Cyclic, branched and branched circular polypeptides mayresult from post-translational natural processes and may be made byentirely synthetic methods, as well.

"Variant(s)" as the term is used herein, is a polynucleotide orpolypeptide that differs from a reference polynucleotide or polypeptiderespectively, but retains essential properties. A typical variant of apolynucleotide differs in nucleotide sequence from another, referencepolynucleotide. Changes in the nucleotide sequence of the variant may ormay not alter the amino acid sequence of a polypeptide encoded by thereference polynucleotide. Nucleotide changes may result in amino acidsubstitutions, additions, deletions, fusions and truncations in thepolypeptide encoded by the reference sequence, as discussed below. Atypical variant of a polypeptide differs in amino acid sequence fromanother, reference polypeptide. Generally, differences are limited sothat the sequences of the reference polypeptide and the variant areclosely similar overall and, in many regions, identical. A variant andreference polypeptide may differ in amino acid sequence by one or moresubstitutions, additions, deletions in any combination. A substituted orinserted amino acid residue may or may not be one encoded by the geneticcode. A variant of a polynucleotide or polypeptide may be a naturallyoccurring such as an allelic variant, or it may be a variant that is notknown to occur naturally. Non-naturally occurring variants ofpolynucleotides and polypeptides may be made by mutagenesis techniques,by direct synthesis, and by other recombinant methods known to skilledartisans.

EXAMPLES

The examples below are carried out using standard techniques, which arewell known and routine to those of skill in the art, except whereotherwise described in detail. The examples are illustrative, but do notlimit the invention.

Example 1 Strain Selection, Library Production and Sequencing

The polynucleotide having a DNA sequence given in Table 1 [SEQ ID NO: 1or 3] was obtained from a library of clones of chromosomal DNA ofStaphylococcus aureus in E. coli. The sequencing data from two or moreclones containing overlapping Staphylococcus aureus DNAs was used toconstruct the contiguous DNA sequence in SEQ ID NO: 1. Libraries may beprepared by routine methods, for example:

Methods 1 and 2 below.

Total cellular DNA is isolated from Staphylococcus aureus WCUH 29according to standard procedures and size-fractionated by either of twomethods.

Method 1

Total cellular DNA is mechanically sheared by passage through a needlein order to size-fractionate according to standard procedures. DNAfragments of up to 11 kbp in size are rendered blunt by treatment withexonuclease and DNA polymerase, and EcoRI linkers added. Fragments areligated into the vector Lambda ZapII that has been cut with EcoRi, thelibrary packaged by standard procedures and E. coli infected with thepackaged library. The library is amplified by standard procedures.

Method 2

Total cellular DNA is partially hydrolyzed with a one or a combinationof restriction enzymes appropriate to generate a series of fragments forcloning into library vectors (e.g., RsaI, Pall, AluI, Bshl235I), andsuch fragments are size-fractionated according to standard procedures.EcoRI linkers are ligated to the DNA and the fragments then ligated intothe vector Lambda ZapII that have been cut with EcoRI, the librarypackaged by standard procedures, and E. coli infected with the packagedlibrary. The library is amplified by standard procedures.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                - (1) GENERAL INFORMATION:                                                    -    (iii) NUMBER OF SEQUENCES: 6                                             - (2) INFORMATION FOR SEQ ID NO:1:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 2599 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                 - ATGTTATTTG GTAGATTAAC TGAACGTGCA CAGCGCGTAT TAGCACATGC AC - #AAGAAGAA         60                                                                          - GCAATTCGTT TAAATCATTC TAATATAGGA ACAGAACACC TATTATTGGG GT - #TAATGAAA        120                                                                          - GAACCTGAAG GAATTGCTGC AAAAGTATTA GAAAGTTTTA ATATCACTGA AG - #ATAAAGTA        180                                                                          - ATTGAAGAAG TTGAAAAATT AATCGGACAT GGTCAAGATC ATGTTGGTAC AT - #TGCATTAT        240                                                                          - ACACCTAGAG CTAAAAAAGT CATTGAATTA TCGATGGATG AAGCTAGAAA AT - #TACATCAC        300                                                                          - AATTTTGTTG GAACGGTTCA TCTTTTATTA GGCTTGATTC GTGAAAATGA AG - #GTGTTGCA        360                                                                          - GCAAGAGTTT TTGCAAATCT AGATTTAAAT ATTACTAAAG CGCGTGCACG GG - #TTGTGAAA        420                                                                          - GCTTTAGGAA ACCCTGAAAT GAGTAATAAA AATGCACAAG CTAGTAAGTC AA - #ATAATACT        480                                                                          - CCAACTTTAG ATAGTTTAGC TCGTGACTTA ACAGTCATTG CCAAAGACGG TA - #CATTAGAT        540                                                                          - CCTGTTATAG GACGTGATAA AGAAATTACA CGTGTAATTG AAGTATTAAG TA - #GACGTACG        600                                                                          - AAAAACAATC CTGTACTTAT TGGAGAGCCA GGTGTTGGTA AAACTGCTAT TG - #CTGAAGGT        660                                                                          - TTAGCGCAAG CCATAGTGAA TAATGAGGTA CCAGAGACAT TAAAAGATAA GC - #GTGTTATG        720                                                                          - TCTTTAGATA TGGGAACAGT AGTTGCAGGT ACTAAATATC GTGGTGAATT TG - #AAGAGCGT        780                                                                          - CTGAAAAAGG TTATGGAAGA AATCCAACAA GCAGGTAATG TCATCCTATT TA - #TTGATGAG        840                                                                          - TTGCATACTT TAGTTGGTGC TGGTGGTGCT GAAGGTGCTA TCGATGCTTC GA - #ATATTTTG        900                                                                          - AAACCGGCAT TAGCACGTGG TGAATTACAA TGTATTGGTG CTACTACATT AG - #ATGAATAT        960                                                                          - CGCAAAAATA TTGAAAAAGA CGCGGCTTTA GAACGTCGTT TCCAACCTGT AC - #AAGTTGAT       1020                                                                          - GAACCTTCAG TAGTAGATAC AGTTGCTATT TTAAAAGGAT TAAGAGATCG TT - #ACGAAGCA       1080                                                                          - CACCATCGTA TTAATATTTC AGACGAAGCT ATTGAAGCAG CTGTTAAATT AA - #GTAACAGA       1140                                                                          - TACGTTTCAG ATCGTTTCTT ACCAGATAAA GCAATTGATT TAATTGATGA AG - #CAAGTTCT       1200                                                                          - AAAGTAAGAC TTAAGAGTCA TACGACACCT AATAATTTAA AAGAAATTGA AC - #AAGAAATT       1260                                                                          - GAAAAAGTTA AAAATGAAAA AGATGCCGCA GTACATGCTC AAGAGTTTGA AA - #ATGCTGCT       1320                                                                          - AACCTGCGTG ATAAACAAAC AAAACTTGAA AAGCAATATG AAGAAGCTAA AA - #ATGAATGG       1380                                                                          - AAGAATGCAC AAAATGGCAT GTCAACTTCA TTGTCAGAAG AAGATATTGC TG - #AAGTTATT       1440                                                                          - GCAGGATGGA CAGGTATCCC ATTAACTAAA ATCAATGAAA CAGAATCTGA AA - #AACTTCTT       1500                                                                          - AGTCTAGAAG ATACATTACA TGAGAGAGTT ATTGGGCAAA AAGATGCTGT TA - #ATTCAATC       1560                                                                          - AGTAAAGCGG TTAGACGTGC CCGTGCAGGG TTAAAAGATC CTAAACGACC AA - #TTGGTAGC       1620                                                                          - TTTATCTTCC TTGGACCAAC TGGTGTTGGT AAAACTGAAT TAGCTAGAGC TT - #TAGCTGAA       1680                                                                          - TCAATGTTTG GCGATGATGA TGCGATGATC CGTGTAGACA TGAGTGAATT TA - #TGGAAAAA       1740                                                                          - CACGCAGTGA GCCGATTAGT TGGTGCTCCT CCAGGATATG TTGGTCATGA TG - #ATGGTGGA       1800                                                                          - CAATTAACTG AAAAAGTTAG ACGTAAACCA TATTCTGTAA TTTTATTTGA TG - #AAATTGAA       1860                                                                          - AAAGCTCATC CAGATGTATT TAATATTCTA TTACAAGTTT TAGATGATGG AC - #ATTTGACA       1920                                                                          - GATACAAAAG GACGTACAGT TGATTTCAGA AATACAATTA TCATAATGAC AT - #CAAACGTT       1980                                                                          - GGGGCACAAG AATTACAAGA TCAACGATTT GCTGGATTCG GTGGTTCAAG TG - #ATGGACAA       2040                                                                          - GATTATGAAA CAATTCGAAA AACGATGTTA AAAGAATTAA AAAATTCATT CC - #GTCCAGAA       2100                                                                          - TTTTTAAACC GTGTAGATGA TATCATTGTA TTCCATAAAC TAACAAAAGA AG - #AATTAAAA       2160                                                                          - GAAATTGTAA CAATGATGGT TAATAAATTA ACAAATCGAT TATCTGAACA AA - #ACATAAAT       2220                                                                          - ATTATTGTTA CTGATAAAGC GAAAGACAAA ATCGCAGAAG AAGGATATGA TC - #CAGAATAT       2280                                                                          - GGTGCAAGAC CATTAATTAG AGCGATACAA AAAACTATCG AAGATAATTT AA - #GTGAATTA       2340                                                                          - ATATTAGATG GTAATCAAAT TGAAGGTAAG AAAGTTACAG TAGATCATGA TG - #GTAAAGAG       2400                                                                          - TTTAAATATG ACATTGCTGA ACAAACTTCA GAAACTAAAA CACCATCGCA AG - #CATAATTA       2460                                                                          - TAAAACAGTC CAAAACAAAT TAAAGTTTTG GGCTGTTTTT TTAGTAGCAT TG - #AACTATAG       2520                                                                          - AAATTCGTGA AAGTATCCAT CAACGAAACA ATCTAATAAA ACAATCATCA AA - #GGATAGTT       2580                                                                          #                 259 - #9                                                    - (2) INFORMATION FOR SEQ ID NO:2:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 866 amino                                                         (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                 - Met Leu Phe Gly Arg Leu Thr Glu Arg Ala Gl - #n Arg Val Leu Ala His         #                15                                                           - Ala Gln Glu Glu Ala Ile Arg Leu Asn His Se - #r Asn Ile Gly Thr Glu         #            30                                                               - His Leu Leu Leu Gly Leu Met Lys Glu Pro Gl - #u Gly Ile Ala Ala Lys         #        45                                                                   - Val Leu Glu Ser Phe Asn Ile Thr Glu Asp Ly - #s Val Ile Glu Glu Val         #    60                                                                       - Glu Lys Leu Ile Gly His Gly Gln Asp His Va - #l Gly Thr Leu His Tyr         #80                                                                           - Thr Pro Arg Ala Lys Lys Val Ile Glu Leu Se - #r Met Asp Glu Ala Arg         #                95                                                           - Lys Leu His His Asn Phe Val Gly Thr Val Hi - #s Leu Leu Leu Gly Leu         #           110                                                               - Ile Arg Glu Asn Glu Gly Val Ala Ala Arg Va - #l Phe Ala Asn Leu Asp         #       125                                                                   - Leu Asn Ile Thr Lys Ala Arg Ala Arg Val Va - #l Lys Ala Leu Gly Asn         #   140                                                                       - Pro Glu Met Ser Asn Lys Asn Ala Gln Ala Se - #r Lys Ser Asn Asn Thr         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Pro Thr Leu Asp Ser Leu Ala Arg Asp Leu Th - #r Val Ile Ala Lys Asp         #               175                                                           - Gly Thr Leu Asp Pro Val Ile Gly Arg Asp Ly - #s Glu Ile Thr Arg Val         #           190                                                               - Ile Glu Val Leu Ser Arg Arg Thr Lys Asn As - #n Pro Val Leu Ile Gly         #       205                                                                   - Glu Pro Gly Val Gly Lys Thr Ala Ile Ala Gl - #u Gly Leu Ala Gln Ala         #   220                                                                       - Ile Val Asn Asn Glu Val Pro Glu Thr Leu Ly - #s Asp Lys Arg Val Met         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Ser Leu Asp Met Gly Thr Val Val Ala Gly Th - #r Lys Tyr Arg Gly Glu         #               255                                                           - Phe Glu Glu Arg Leu Lys Lys Val Met Glu Gl - #u Ile Gln Gln Ala Gly         #           270                                                               - Asn Val Ile Leu Phe Ile Asp Glu Leu His Th - #r Leu Val Gly Ala Gly         #       285                                                                   - Gly Ala Glu Gly Ala Ile Asp Ala Ser Asn Il - #e Leu Lys Pro Ala Leu         #   300                                                                       - Ala Arg Gly Glu Leu Gln Cys Ile Gly Ala Th - #r Thr Leu Asp Glu Tyr         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - Arg Lys Asn Ile Glu Lys Asp Ala Ala Leu Gl - #u Arg Arg Phe Gln Pro         #               335                                                           - Val Gln Val Asp Glu Pro Ser Val Val Asp Th - #r Val Ala Ile Leu Lys         #           350                                                               - Gly Leu Arg Asp Arg Tyr Glu Ala His His Ar - #g Ile Asn Ile Ser Asp         #       365                                                                   - Glu Ala Ile Glu Ala Ala Val Lys Leu Ser As - #n Arg Tyr Val Ser Asp         #   380                                                                       - Arg Phe Leu Pro Asp Lys Ala Ile Asp Leu Il - #e Asp Glu Ala Ser Ser         385                 3 - #90                 3 - #95                 4 -       #00                                                                           - Lys Val Arg Leu Lys Ser His Thr Thr Pro As - #n Asn Leu Lys Glu Ile         #               415                                                           - Glu Gln Glu Ile Glu Lys Val Lys Asn Glu Ly - #s Asp Ala Ala Val His         #           430                                                               - Ala Gln Glu Phe Glu Asn Ala Ala Asn Leu Ar - #g Asp Lys Gln Thr Lys         #       445                                                                   - Leu Glu Lys Gln Tyr Glu Glu Ala Lys Asn Gl - #u Trp Lys Asn Ala Gln         #   460                                                                       - Asn Gly Met Ser Thr Ser Leu Ser Glu Glu As - #p Ile Ala Glu Val Ile         465                 4 - #70                 4 - #75                 4 -       #80                                                                           - Ala Gly Trp Thr Gly Ile Pro Leu Thr Lys Il - #e Asn Glu Thr Glu Ser         #               495                                                           - Glu Lys Leu Leu Ser Leu Glu Asp Thr Leu Hi - #s Glu Arg Val Ile Gly         #           510                                                               - Gln Lys Asp Ala Val Asn Ser Ile Ser Lys Al - #a Val Arg Arg Ala Arg         #       525                                                                   - Ala Gly Leu Lys Asp Pro Lys Arg Pro Ile Gl - #y Ser Phe Ile Phe Leu         #   540                                                                       - Gly Pro Thr Gly Val Gly Lys Thr Glu Leu Al - #a Arg Ala Leu Ala Glu         545                 5 - #50                 5 - #55                 5 -       #60                                                                           - Ser Met Phe Gly Asp Asp Asp Ala Met Ile Ar - #g Val Asp Met Ser Glu         #               575                                                           - Phe Met Glu Lys His Ala Val Ser Arg Leu Va - #l Gly Ala Pro Pro Gly         #           590                                                               - Tyr Val Gly His Asp Asp Gly Gly Gln Leu Th - #r Glu Lys Val Arg Arg         #       605                                                                   - Lys Pro Tyr Ser Val Ile Leu Phe Asp Glu Il - #e Glu Lys Ala His Pro         #   620                                                                       - Asp Val Phe Asn Ile Leu Leu Gln Val Leu As - #p Asp Gly His Leu Thr         625                 6 - #30                 6 - #35                 6 -       #40                                                                           - Asp Thr Lys Gly Arg Thr Val Asp Phe Arg As - #n Thr Ile Ile Ile Met         #               655                                                           - Thr Ser Asn Val Gly Ala Gln Glu Leu Gln As - #p Gln Arg Phe Ala Gly         #           670                                                               - Phe Gly Gly Ser Ser Asp Gly Gln Asp Tyr Gl - #u Thr Ile Arg Lys Thr         #       685                                                                   - Met Leu Lys Glu Leu Lys Asn Ser Phe Arg Pr - #o Glu Phe Leu Asn Arg         #   700                                                                       - Val Asp Asp Ile Ile Val Phe His Lys Leu Th - #r Lys Glu Glu Leu Lys         705                 7 - #10                 7 - #15                 7 -       #20                                                                           - Glu Ile Val Thr Met Met Val Asn Lys Leu Th - #r Asn Arg Leu Ser Glu         #               735                                                           - Gln Asn Ile Asn Ile Ile Val Thr Asp Lys Al - #a Lys Asp Lys Ile Ala         #           750                                                               - Glu Glu Gly Tyr Asp Pro Glu Tyr Gly Ala Ar - #g Pro Leu Ile Arg Ala         #       765                                                                   - Ile Gln Lys Thr Ile Glu Asp Asn Leu Ser Gl - #u Leu Ile Leu Asp Gly         #   780                                                                       - Asn Gln Ile Glu Gly Lys Lys Val Thr Val As - #p His Asp Gly Lys Glu         785                 7 - #90                 7 - #95                 8 -       #00                                                                           - Phe Lys Tyr Asp Ile Ala Glu Gln Thr Ser Gl - #u Thr Lys Thr Pro Ser         #               815                                                           - Gln Ala Xaa Leu Xaa Asn Ser Pro Lys Gln Il - #e Lys Val Leu Gly Cys         #           830                                                               - Phe Phe Ser Ser Ile Glu Leu Xaa Lys Phe Va - #l Lys Val Ser Ile Asn         #       845                                                                   - Glu Thr Ile Xaa Xaa Asn Asn His Gln Arg Il - #e Val Lys Asn Tyr Met         #   860                                                                       - Xaa Gln                                                                     865                                                                           - (2) INFORMATION FOR SEQ ID NO:3:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 2019 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                 - ATGAGTAATA AAAATGCACA AGCTAGTAAG TCAAATAATA CTCCAACTTT AG - #ATAGTTTA         60                                                                          - GCTCGTGACT TAACAGTCAT TGCCAAAGAC GGTACATTAG ATCCTGTTAT AG - #GACGTGAT        120                                                                          - AAAGAAATTA CACGTGTAAT TGAAGTATTA AGTAGACGTA CGAAAAACAA TC - #CTGTACTT        180                                                                          - ATTGGAGAGC CAGGTGTTGG TAAAACTGCT ATTGCTGAAG GTTTAGCGCA AG - #CCATAGTG        240                                                                          - AATAATGAGG TACCAGAGAC ATTAAAAGAT AAGCGTGTTA TGTCTTTAGA TA - #TGGGAACA        300                                                                          - GTAGTTGCAG GTACTAAATA TCGTGGTGAA TTTGAAGAGC GTCTGAAAAA GG - #TTATGGAA        360                                                                          - GAAATCCAAC AAGCAGGTAA TGTCATCCTA TTTATTGATG AGTTGCATAC TT - #TAGTTGGT        420                                                                          - GCTGGTGGTG CTGAAGGTGC TATCGATGCT TCGAATATTT TGAAACCGGC AT - #TAGCACGT        480                                                                          - GGTGAATTAC AATGTATTGG TGCTACTACA TTAGATGAAT ATCGCAAAAA TA - #TTGAAAAA        540                                                                          - GACGCGGCTT TAGAACGTCG TTTCCAACCT GTACAAGTTG ATGAACCTTC AG - #TAGTAGAT        600                                                                          - ACAGTTGCTA TTTTAAAAGG ATTAAGAGAT CGTTACGAAG CACACCATCG TA - #TTAATATT        660                                                                          - TCAGACGAAG CTATTGAAGC AGCTGTTAAA TTAAGTAACA GATACGTTTC AG - #ATCGTTTC        720                                                                          - TTACCAGATA AAGCAATTGA TTTAATTGAT GAAGCAAGTT CTAAAGTAAG AC - #TTAAGAGT        780                                                                          - CATACGACAC CTAATAATTT AAAAGAAATT GAACAAGAAA TTGAAAAAGT TA - #AAAATGAA        840                                                                          - AAAGATGCCG CAGTACATGC TCAAGAGTTT GAAAATGCTG CTAACCTGCG TG - #ATAAACAA        900                                                                          - ACAAAACTTG AAAAGCAATA TGAAGAAGCT AAAAATGAAT GGAAGAATGC AC - #AAAATGGC        960                                                                          - ATGTCAACTT CATTGTCAGA AGAAGATATT GCTGAAGTTA TTGCAGGATG GA - #CAGGTATC       1020                                                                          - CCATTAACTA AAATCAATGA AACAGAATCT GAAAAACTTC TTAGTCTAGA AG - #ATACATTA       1080                                                                          - CATGAGAGAG TTATTGGGCA AAAAGATGCT GTTAATTCAA TCAGTAAAGC GG - #TTAGACGT       1140                                                                          - GCCCGTGCAG GGTTAAAAGA TCCTAAACGA CCAATTGGTA GCTTTATCTT CC - #TTGGACCA       1200                                                                          - ACTGGTGTTG GTAAAACTGA ATTAGCTAGA GCTTTAGCTG AATCAATGTT TG - #GCGATGAT       1260                                                                          - GATGCGATGA TCCGTGTAGA CATGAGTGAA TTTATGGAAA AACACGCAGT GA - #GCCGATTA       1320                                                                          - GTTGGTGCTC CTCCAGGATA TGTTGGTCAT GATGATGGTG GACAATTAAC TG - #AAAAAGTT       1380                                                                          - AGACGTAAAC CATATTCTGT AATTTTATTT GATGAAATTG AAAAAGCTCA TC - #CAGATGTA       1440                                                                          - TTTAATATTC TATTACAAGT TTTAGATGAT GGACATTTGA CAGATACAAA AG - #GACGTACA       1500                                                                          - GTTGATTTCA GAAATACAAT TATCATAATG ACATCAAACG TTGGGGCACA AG - #AATTACAA       1560                                                                          - GATCAACGAT TTGCTGGATT CGGTGGTTCA AGTGATGGAC AAGATTATGA AA - #CAATTCGA       1620                                                                          - AAAACGATGT TAAAAGAATT AAAAAATTCA TTCCGTCCAG AATTTTTAAA CC - #GTGTAGAT       1680                                                                          - GATATCATTG TATTCCATAA ACTAACAAAA GAAGAATTAA AAGAAATTGT AA - #CAATGATG       1740                                                                          - GTTAATAAAT TAACAAATCG ATTATCTGAA CAAAACATAA ATATTATTGT TA - #CTGATAAA       1800                                                                          - GCGAAAGACA AAATCGCAGA AGAAGGATAT GATCCAGAAT ATGGTGCAAG AC - #CATTAATT       1860                                                                          - AGAGCGATAC AAAAAACTAT CGAAGATAAT TTAAGTGAAT TAATATTAGA TG - #GTAATCAA       1920                                                                          - ATTGAAGGTA AGAAAGTTAC AGTAGATCAT GATGGTAAAG AGTTTAAATA TG - #ACATTGCT       1980                                                                          #  2019            CTAA AACACCATCG CAAGCATAA                                  - (2) INFORMATION FOR SEQ ID NO:4:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 672 amino                                                         (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                  - Met Ser Asn Lys Asn Ala Gln Ala Ser Lys Se - #r Asn Asn Thr Pro Thr         #                15                                                           - Leu Asp Ser Leu Ala Arg Asp Leu Thr Val Il - #e Ala Lys Asp Gly Thr         #            30                                                               - Leu Asp Pro Val Ile Gly Arg Asp Lys Glu Il - #e Thr Arg Val Ile Glu         #        45                                                                   - Val Leu Ser Arg Arg Thr Lys Asn Asn Pro Va - #l Leu Ile Gly Glu Pro         #    60                                                                       - Gly Val Gly Lys Thr Ala Ile Ala Glu Gly Le - #u Ala Gln Ala Ile Val         #80                                                                           - Asn Asn Glu Val Pro Glu Thr Leu Lys Asp Ly - #s Arg Val Met Ser Leu         #                95                                                           - Asp Met Gly Thr Val Val Ala Gly Thr Lys Ty - #r Arg Gly Glu Phe Glu         #           110                                                               - Glu Arg Leu Lys Lys Val Met Glu Glu Ile Gl - #n Gln Ala Gly Asn Val         #       125                                                                   - Ile Leu Phe Ile Asp Glu Leu His Thr Leu Va - #l Gly Ala Gly Gly Ala         #   140                                                                       - Glu Gly Ala Ile Asp Ala Ser Asn Ile Leu Ly - #s Pro Ala Leu Ala Arg         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Gly Glu Leu Gln Cys Ile Gly Ala Thr Thr Le - #u Asp Glu Tyr Arg Lys         #               175                                                           - Asn Ile Glu Lys Asp Ala Ala Leu Glu Arg Ar - #g Phe Gln Pro Val Gln         #           190                                                               - Val Asp Glu Pro Ser Val Val Asp Thr Val Al - #a Ile Leu Lys Gly Leu         #       205                                                                   - Arg Asp Arg Tyr Glu Ala His His Arg Ile As - #n Ile Ser Asp Glu Ala         #   220                                                                       - Ile Glu Ala Ala Val Lys Leu Ser Asn Arg Ty - #r Val Ser Asp Arg Phe         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Leu Pro Asp Lys Ala Ile Asp Leu Ile Asp Gl - #u Ala Ser Ser Lys Val         #               255                                                           - Arg Leu Lys Ser His Thr Thr Pro Asn Asn Le - #u Lys Glu Ile Glu Gln         #           270                                                               - Glu Ile Glu Lys Val Lys Asn Glu Lys Asp Al - #a Ala Val His Ala Gln         #       285                                                                   - Glu Phe Glu Asn Ala Ala Asn Leu Arg Asp Ly - #s Gln Thr Lys Leu Glu         #   300                                                                       - Lys Gln Tyr Glu Glu Ala Lys Asn Glu Trp Ly - #s Asn Ala Gln Asn Gly         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - Met Ser Thr Ser Leu Ser Glu Glu Asp Ile Al - #a Glu Val Ile Ala Gly         #               335                                                           - Trp Thr Gly Ile Pro Leu Thr Lys Ile Asn Gl - #u Thr Glu Ser Glu Lys         #           350                                                               - Leu Leu Ser Leu Glu Asp Thr Leu His Glu Ar - #g Val Ile Gly Gln Lys         #       365                                                                   - Asp Ala Val Asn Ser Ile Ser Lys Ala Val Ar - #g Arg Ala Arg Ala Gly         #   380                                                                       - Leu Lys Asp Pro Lys Arg Pro Ile Gly Ser Ph - #e Ile Phe Leu Gly Pro         385                 3 - #90                 3 - #95                 4 -       #00                                                                           - Thr Gly Val Gly Lys Thr Glu Leu Ala Arg Al - #a Leu Ala Glu Ser Met         #               415                                                           - Phe Gly Asp Asp Asp Ala Met Ile Arg Val As - #p Met Ser Glu Phe Met         #           430                                                               - Glu Lys His Ala Val Ser Arg Leu Val Gly Al - #a Pro Pro Gly Tyr Val         #       445                                                                   - Gly His Asp Asp Gly Gly Gln Leu Thr Glu Ly - #s Val Arg Arg Lys Pro         #   460                                                                       - Tyr Ser Val Ile Leu Phe Asp Glu Ile Glu Ly - #s Ala His Pro Asp Val         465                 4 - #70                 4 - #75                 4 -       #80                                                                           - Phe Asn Ile Leu Leu Gln Val Leu Asp Asp Gl - #y His Leu Thr Asp Thr         #               495                                                           - Lys Gly Arg Thr Val Asp Phe Arg Asn Thr Il - #e Ile Ile Met Thr Ser         #           510                                                               - Asn Val Gly Ala Gln Glu Leu Gln Asp Gln Ar - #g Phe Ala Gly Phe Gly         #       525                                                                   - Gly Ser Ser Asp Gly Gln Asp Tyr Glu Thr Il - #e Arg Lys Thr Met Leu         #   540                                                                       - Lys Glu Leu Lys Asn Ser Phe Arg Pro Glu Ph - #e Leu Asn Arg Val Asp         545                 5 - #50                 5 - #55                 5 -       #60                                                                           - Asp Ile Ile Val Phe His Lys Leu Thr Lys Gl - #u Glu Leu Lys Glu Ile         #               575                                                           - Val Thr Met Met Val Asn Lys Leu Thr Asn Ar - #g Leu Ser Glu Gln Asn         #           590                                                               - Ile Asn Ile Ile Val Thr Asp Lys Ala Lys As - #p Lys Ile Ala Glu Glu         #       605                                                                   - Gly Tyr Asp Pro Glu Tyr Gly Ala Arg Pro Le - #u Ile Arg Ala Ile Gln         #   620                                                                       - Lys Thr Ile Glu Asp Asn Leu Ser Glu Leu Il - #e Leu Asp Gly Asn Gln         625                 6 - #30                 6 - #35                 6 -       #40                                                                           - Ile Glu Gly Lys Lys Val Thr Val Asp His As - #p Gly Lys Glu Phe Lys         #               655                                                           - Tyr Asp Ile Ala Glu Gln Thr Ser Glu Thr Ly - #s Thr Pro Ser Gln Ala         #           670                                                               - (2) INFORMATION FOR SEQ ID NO:5:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 26 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                 #              26  CTAC TGAAGG                                                - (2) INFORMATION FOR SEQ ID NO:6:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 26 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                 #              26  CTAC TGAAGG                                                __________________________________________________________________________

What is claimed is:
 1. An isolated polynucleotide segment comprising afirst polynucleotide sequence or the full complement of the entirelength of the first polynucleotide sequence, wherein the firstpolynucleotide sequence encodes a polypeptide comprising the amino acidsequence set forth in SEQ ID NO:2.
 2. The isolated polynucleotidesegment of claim 1, wherein the isolated polynucleotide segmentcomprises the first polynucleotide sequence.
 3. A vector comprising theisolated polynucleotide segment of claim
 2. 4. An isolated host cellcomprising the vector of claim
 3. 5. A process for producing apolypeptide comprising the step of culturing the host cell of claim 4under conditions sufficient for the production of the polypeptide,wherein the polypeptide is encoded by the first polynucleotide sequence.6. The isolated polynucleotide segment of claim 1, wherein the isolatedpolynucleotide segment comprises the full complement of the entirelength of the first polynucleotide sequence.
 7. A vector comprising theisolated polynucleotide segment of claim
 6. 8. An isolated host cellcomprising the vector of claim
 7. 9. A polynucleotide which encodes afusion polypeptide and which includes the isolated polynucleotidesegment according to claim
 2. 10. An isolated polynucleotide segmentcomprising a first polynucleotide sequence or the full complement of theentire length of the first polynucleotide sequence, wherein the firstpolynucleotide sequence is identical to SEQ ID NO: 1, except that, overthe entire length corresponding to SEQ ID NO:1, n_(n) nucleotides aresubstituted, inserted or deleted, wherein n_(n) satisfies the followingexpression

    n.sub.n ≦x.sub.n -(x.sub.n ·y)

wherein x_(n) is the total number of nucleotides in SEQ ID NO: 1, y isat least 0.998, and wherein any non-integer product of x_(n) and y isrounded down to the nearest integer before subtracting the product fromx_(n).
 11. A vector comprising the isolated polynucleotide segment ofclaim
 10. 12. An isolated host cell comprising the vector of claim 11.13. An isolated polynucleotide segment comprising a first polynucleotidesequence or the full complement of the entire length of the firstpolynucleotide sequence, wherein the first polynucleotide sequencecomprises SEQ ID NO:
 1. 14. The isolated polynucleotide segment of claim13, wherein the isolated polynucleotide segment comprises the firstpolynucleotide sequence.
 15. A vector comprising the isolatedpolynucleotide segment of claim
 14. 16. An isolated host cell comprisingthe vector of claim
 15. 17. A process for producing a polypeptidecomprising the step of culturing the host cell of claim 16 underconditions sufficient for the production of the polypeptide, wherein thepolypeptide is encoded by the first polynucleotide sequence.
 18. Theisolated polynucleotide segment of claim 13, wherein the isolatedpolynucleotide segment comprises the full complement of the entirelength of the first polynucleotide sequence.
 19. A vector comprising theisolated polynucleotide segment of claim
 18. 20. An isolated host cellcomprising the vector of claim
 19. 21. A polynucleotide which encodes afusion polypeptide and which includes the isolated polynucleotidesegment according to claim
 13. 22. An isolated polynucleotide segmentcomprising a first polynucleotide sequence or the full complement of theentire length of the first polynucleotide sequence, wherein the firstpolynucleotide sequence encodes a polypeptide consisting of the aminoacid sequence set forth in SEQ ID NO:2.
 23. The isolated polynucleotidesegment of claim 22, wherein the isolated polynucleotide segmentcomprises the first polynucleotide sequence.
 24. A vector comprising theisolated polynucleotide segment of claim
 23. 25. An isolated host cellcomprising the vector of claim
 24. 26. A process for producing apolypeptide comprising the step of culturing the host cell of claim 25under conditions sufficient for the production of the polypeptide,wherein the polypeptide is encoded by the first polynucleotide sequence.27. The isolated polynucleotide segment of claim 22, wherein theisolated polynucleotide segment comprises the full complement of theentire length of the first polynucleotide sequence.
 28. A vectorcomprising the isolated polynucleotide segment of claim
 27. 29. Anisolated host cell comprising the vector of claim 28.